Processes for conversion of biologically derived mevalonic acid

ABSTRACT

The invention relates to a process comprising reacting mevalonic acid, or a solution comprising mevalonic acid, to yield a first product or first product mixture, optionally in the presence of a solid catalyst and/or at elevated temperature and/or pressure. The invention further relates to a process comprising: (a) providing a microbial organism that expresses a biosynthetic mevalonic acid pathway; (b) growing the microbial organism in fermentation medium comprising suitable carbon substrates, whereby biobased mevalonic acid is produced; and (c) reacting said biobased mevalonic acid to yield a first product or first product mixture.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the priority benefit of U.S. Provisional Patent Application Ser. No. 62/084,689, filed Nov. 26, 2014, entitled Processes for Conversion of Biologically Derived Mevalonic Acid, incorporated by reference in its entirety herein.

SEQUENCE LISTING

The following application contains a sequence listing in computer readable format (CRF), submitted as a text file in ASCII format entitled “46567SequenceListing,” created on Nov. 23, 2015, as 25 KB. The content of the CRF is hereby incorporated by reference.

BACKGROUND OF INVENTION

1. Technical Field

The present invention relates generally to processes for producing intermediates for hydrocarbon biofuels and other products from organic acids that are derived from via biochemical routes. In particular, Mevalonic acid (or Mevalonolactone) can be derived from biomass fermentation to produce a variety of intermediates. One or more embodiments of the present invention relate to production of isoprene, methyl vinyl ketone, 3-methyl-2-butanone, and anhydro-mevalonolactone.

2. Description of Related Art

Organic acids are an important class of compounds that can be derived via biochemical routes and can serve as intermediates for the sustainable production of hydrocarbon biofuels and other products. Of particular interest is the dehydration/decarboxylation to form isoprene, a critical starting material for a variety of synthetic polymers (i.e., synthetic rubbers), specialty chemicals, and a gasoline additive, known as alkylate. While isoprene can be obtained by fractionating petroleum, the purification of this material is expensive and time-consuming. In addition, the depletion of fossil fuels makes it crucial that another source of this vital starting material be found. Isoprene is naturally produced by a variety of microbial, plant, and animal species. However, the yield of isoprene from naturally-occurring organisms is commercially unattractive. Thus, a large scale synthesis of isoprene, and other synthetically useful intermediates, from biomass-derived mevalonolactone would have substantial commercial potential.

SUMMARY OF THE INVENTION

In one aspect, the invention is concerned with a process comprising: (a) reacting MVL, or a solution comprising MVL, in the presence of a solid catalyst to yield a first product or first product mixture, wherein the solution comprising MVL optionally comprises anhydro-mevalonolactone and/or a co-reactant.

In some embodiments the co-reactant is selected from the group consisting of water, alkyl alcohol, ethers, aromatic compounds, acids, aldehydes, esters, and mixtures thereof. In some embodiments, step (a) occurs at a temperature in the range of about 100° C. to about 500° C. In some embodiments, step (a) occurs at a pressure in the range of about sub-atmospheric pressure to about 200 bar.

In some embodiments the solid catalyst is a solid acid catalyst. A non-limiting, illustrative list of potentially suitable solid catalysts includes functionalized styrene-divinylbenzene copolymers, functionalized tetrafluoroethylene-fluoropolymer copolymers, calcium apatite, silica-alumina, silica, titania, alumina, resins, metal oxides, and/or zeolites.

In some embodiments the solid catalyst is selected from the group consisting of a metal oxide catalyst and a carbon catalyst. In some embodiments the solid catalyst has a surface area between about 20 m²/g and about 600 m²/g. In some embodiments of the solid catalyst is selected from the group consisting of silicon oxide, aluminum oxide, titanium oxide, magnesium oxide, zirconium oxide, cobalt oxide, iron oxide, nickel oxide, manganese oxide, zinc oxide, molybdenum oxide, tungsten oxide, calcium oxide, cerium oxide, tin oxide, and chromium oxide, and mixtures thereof. In some embodiments the solid catalyst comprises a mixed metal oxide catalyst.

In some embodiments of the invention the solid catalyst comprises a supported transition or noble metal, e.g., palladium, magnesium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, zirconium, molybdenum, ruthenium, rhodium, silver, tin, tungsten, tantalum, iridium, platinum, and gold, and mixtures thereof.

In some embodiments, the first product or first product mixture comprises one or more of isoprene, 3-methyl-2-butanone, methyl vinyl ketone, anhydro-mevalonolactone, 2-pentene, 2-methyl-1-butene, 2-methyl-1-pentene, methane, hydrogen, or an alkyl alcohol.

In some embodiments of the invention, the previously described process comprises an additional step of: (b) reacting said first product or first product mixture with a first reagent and a second catalyst to yield a second product or second product mixture, wherein said first reagent is water or an olefin or an alcohol. In some embodiments the first reagent is an olefin and said second catalyst is selected from the group consisting of palladium, platinum, ruthenium, molybdenum, tungsten, rhenium, tin, and rhodium olefin metathesis catalysts, and mixtures thereof. In some embodiments the first reagent is an alcohol and said second catalyst is selected from the group consisting of homogenous and heterogeneous acids and bases, sugars, lipases, ion exchange resins, metal oxides, and zeolites, and mixtures thereof.

In some embodiments, the previously described step (b) occurs at a temperature in the range of about 100° C. to about 500° C. In some embodiments this process further comprises a step of: (c) reacting said second product or second product mixture with a second reagent and a third catalyst to yield a third product or third product mixture.

In another aspect, the invention provides a process comprising: (a) reacting MVL, or a solution comprising MVL, at a temperature in the range of about 100° C. to about 500° C. to yield a first product or first product mixture.

And yet another aspect, the invention provides a process comprising: (a) providing a microbial organism that expresses a biosynthetic MVL pathway; (b) growing the microbial organism in fermentation medium comprising suitable carbon substrates, whereby biobased MVL is produced; and (c) reacting said biobased MVL to yield a first product or first product mixture. Step (c) optionally occurs in the presence of a solid catalyst and/or at a temperature in the range of about 100° C. to about 500° C.

In some embodiments, a microbial organism used in the above described process is a non-naturally occurring microbial organism comprising a metabolic modification that enables biobased MVL production.

In some embodiments, the metabolic modifications comprises expression of at least one gene set selected from the group consisting of: i. mvaE, mvaS; ii. mvaE, mvaS, atoB; iii. atoB, hmgR, hmgS; and iv. functional homologs of i, ii, or iii.

In some embodiments, the metabolic modification comprises modification of at least one gene, e.g., ackA-pta, adhE, and/or ldhA.

In some embodiments, the fermentation medium used comprises a renewable feedstock.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 (FIG. 1) is a schematic diagram of a high pressure small scale unit used in Example 11.

DETAILED DESCRIPTION

Mevalonic acid, mevalonate, and mevalonolactone exist in an equilibrium which is pH dependent. Thus, a solution which contains “mevalonate” may actually contain mevalonic acid, mevalonate, and/or mevalonolactone. In addition, depending on the pH, the components may be in salt form. Depending on the reagent chosen, the counter ion can be the cation of ammonium, sodium, lithium, potassium, magnesium, calcium, aluminum, or cesium. For convenience, mevalonolactone or “MVL” will be used herein inclusively.

Mevalonolactone (MVL) and its related derivatives, such as 2,3-dehydromevalonolactone (4-methyl-5,6-dihydro-2H-pyran-2-one; also referred to as anhydromevalonolactone, AML) represent a potentially abundant feedstock that may be prepared on an industrial scale. Biobased production via fermentation allows for the high-yielding and cost competitive production of MVL from renewable feedstocks with low volatility, such as sugars, glycerin, syngas, or methane. Chemical products produced from biobased MVL and its related derivatives could fill a need for inexpensive, renewable consumer and industrial products not based on petroleum or other nonrenewable resources. Biobased mevalonolactone can subsequently be converted into a variety of useful compounds. Exemplary compounds are described herein, and also in co-pending U.S. Ser. No. 14/939,437, filed Nov. 12, 2015, incorporated by reference herein in its entirety.

The term “biobased” as used herein means that the compound was synthesized from a biological precursor, and specifically a renewable biological carbon source, such as biomass (as opposed to a non-renewable petroleum-based carbon source). ASTM has set a method standard to calculate the level of biobased material included in a composition: ASTM D6866—Standard Test Methods for Determining the Biobased Content of Solid, Liquid, and Gaseous Samples Using Radiocarbon Analysis. The biobased content of a composition is the amount of biobased carbon in the material as fraction weight (mass) or percent weight (mass) of the total organic carbon in the material. ASTM D6866 is similar to radiocarbon dating without the age equations. It is done by determining a ratio of the amount of radiocarbon (14C) in the material to that of a modern reference standard. The ratio is reported as a percentage referred to as percent modern carbon (units “pMC”). If the material being analyzed is a mixture of present day radiocarbon and fossil carbon (i.e., containing no radiocarbon), then the pMC value obtained correlates directly to the amount of biobased material present in the sample.

ASTM D6866 distinguishes carbon resulting from contemporary biomass-based input material from those derived from fossil-based material. Biomass contains a well-characterized amount of Carbon-14 that is easily differentiated from other materials such as fossil fuels that do not contain any Carbon-14. “Biomass” is generally defined as plant material, vegetation or agricultural waste used as a fuel or energy source. The ratio of carbon-14 isotope to carbon-12 isotope for biomass carbon is generally known to those skilled in the art to be about 2×10⁻¹² to 1 based on the current natural abundance of carbon-14 to carbon-12 as taken from air samples. Since the amount of Carbon-14 in biomass is known, a percentage of carbon from renewable sources can be calculated easily from the total organic carbon in the sample. Zero percent Carbon-14 indicates a complete lack of Carbon-14 atoms in a material, thus indicating a fossil or petroleum-based carbon source. Likewise, 100% Carbon-14 (after atmospheric correction) indicates a modern biobased carbon source.

The modern reference standard used in radiocarbon dating is a NIST (National Institute of Standards and Technology) standard with a known radiocarbon content equivalent approximately to the year AD 1950. AD 1950 was chosen since it represented a time prior to thermo-nuclear weapons testing which introduced large amounts of excess radiocarbon into the atmosphere with each explosion (termed “bomb carbon”). This was a logical point in time to use as a reference for archaeologists and geologists. For reference, an archaeologist or geologist using radiocarbon dates, AD 1950 equals “zero years old.” It also represents 100 pMC. “Bomb carbon” in the atmosphere reached almost twice normal levels in 1963 at the peak of testing and prior to the treaty halting the testing. Its distribution within the atmosphere has been approximated since its appearance, showing values that are greater than 100 pMC for plants and animals living since AD 1950. It has gradually decreased over time with today's value being near 105 pMC. This means that a fresh biomass material such as corn, sugar cane or soybeans would give a radiocarbon signature near 105 pMC. By presuming ˜105 pMC represents present day biomass materials and 0 pMC represents petroleum derivatives, the measured pMC value for that material will reflect the proportions of the two component types. For example, a material derived 100% from present day soybeans would give a radiocarbon signature near 105 pMC. But if it was diluted with 50% petroleum carbon, it would give a radiocarbon signature near 53 pMC.

The “biobased content” of a material is reported as a percent value relating total renewable organic carbon to total organic carbon. The final result is calculated by multiplying the pMC value measured for the material by 0.95 (to adjust for bomb carbon effect). The final % value is cited as the mean biobased result and assumes all the components within the analyzed material were either present day living (within the last decade) or fossil in origin. In one aspect, the materials used in the invention (e.g., precursor compounds or resulting polymers) have a biobased content of greater than 0%, more preferably greater than 10%, more preferably greater than 25%, more preferably greater than 50%, and even more preferably greater than 75%. Preferably, the materials used in the invention are substantially entirely biobased according to ASTM D6866, which means they are 95 percent or more of biological origin. Thus, it will be appreciated that biobased products can be distinguished from petroleum-based products by carbon finger-printing. Thus, biobased polymers and polymeric precursors according to the invention will have a higher radioactive Carbon-14 (14C) content or a higher 14C/12C ratio than the same type of polymer of petroleum (non-renewable) origin. In one aspect, the biobased polymeric precursors and/or resulting polymers will have a 14C/12C ratio of greater than 0, preferably greater than 1.

The biobased MVL can serve as a common starting material for the production of several classes of polymers, as described in co-pending US S/N 14/939,437.

For example, MVL and AML themselves are monomers for polymerization reactions. The lactone structure of MVL and AML allows for ring-opening co-polymerization. The ring-opening co-polymerization of MVL-derived 13-methyl-6-valerolactone has been reported previously. In addition to ring-opening polymerization, AML may be a co-monomer for free radical polymerization reactions. During the dehydration reaction of MVL different AML isomers, differing by the position of the unsaturation, i.e., 3,4-dehydromevalonolactone (4-methyl-3,6-dihydro-2H-pyran-2-one), 4,5-dehydromevalonolactone (4-methyl-3,4-dihydro-2H-pyran-2-one), and exo-dehydromevalonolactone (4-methylenetetrahydro-2H-pyran-2-one), can be obtained. These isomers may be utilized as monomers for ring-opening and free radical polymerization similar to AML.

In addition to the direct polymerization of MVL and related compounds described above, they can be converted into building blocks for step-growth polymers, such as diols and diacids among others. These conversions can be broadly categorized by the type of chemical reaction: i) Oxidation, ii) Reduction, iii) Lactone ring-opening, iv) Olefin modification, and v) Alcohol modification.

Full oxidation of MVL and related lactones to afford alpha,omega-dicarboxylic acids can be realized by treatment with high-valent metal oxides such as Cr06, or KMnO₄ among others, which can also be employed in catalytic amounts in the presence of a stoichiometric strong oxidant. The oxidation of primary alcohols to carboxylic acids is also possible with nitric acid, or molecular oxygen as the oxidant. Examples of above reagents can be found for carboxylic acid as well as lactone oxidation. The MVL-derived dicarboxylic acids may find use as building blocks for polyesters, alkyd resins, unsaturated polyester resins, polyester polyols, or polyamides to name a few.

Full reduction of MVL and related lactones leads to substituted alpha,omega-diols. Metal hydrides, such as lithium tetrahydridoaluminate, elemental sodium or hydrogen in the presence of a metal catalyst can be employed to reduce the lactone functional group to two primary alcohols. These MVL-derived di-, and polyols may be used as building blocks for polyesters, alkyd resins, unsaturated polyester resins, polyester polyols, polycarbonates, or vinyl-urethane resins among others.

Nucleophiles such as alcohols, thiols, or amines are able to ring-open lactones. The reaction of MVL and related lactones with di-, or multifunctional nucleophiles results in the formation of substituted ring-opened MVL-based di-, or polyols. The nucleophiles can be either homofunctional, e.g. glycerol, or ethylene diamine among others, or heterofunctional, such as ethanolamine, or mercapto ethanol among others. The uses of these ring-opened derivatives are equivalent to the above mentioned MVL reduction products.

Modifications of the alkene group in AML and its isomers include epoxidation, dihydroxylation, Michael addition, Diels-Alder reaction, or [2+2]-cycloaddition among others. The reaction products are diverse in functionality, and possibly serve as building blocks for a variety of polymers. The lactone functionality is preserved in all the above mentioned reactions. Ring-opening polymerization of these compounds can result in the formation of modified aliphatic polyester with applications as thermoplastic elastomers, unsaturated polyester resins, or polyurethanes among others.

The tertiary alcohol in MVL can be modified to form ether and ester derivatives. Functional groups promoting polymerization, or properties such as solubility and adhesion can be introduced. It has been reported that a methacrylate with pendant MVL ester group can be obtained from MVL. It can be envisioned that this bi-functional adduct is able to participate in free-radical and ring-opening polymerization to produce acrylics and vinyl polyesters.

The MVL-derived di-, or polyols and -acids can also serve as precursors for epoxy resins, poly(vinylether)s, or multifunctional cyclic carbonates.

In some embodiments, the invention provides a process that comprises a first step of reacting MVL, or a solution comprising MVL, under conditions that yield a first product or first product mixture. In an optional second step, the first product or first product mixture can then be further reacted with a reagent, optionally in the presence of a catalyst. In some embodiments of the invention, the reagent is an olefin or an alcohol. In an optional third step, the second product or second product mixture resulting from this reaction can be reacted with the same or another reagent, again optionally in the presence of a catalyst to produce a third product or third product mixture. As used herein, the term “product mixture” refers to a mixture of structurally distinct compounds.

In certain aspects of the invention, a product or product mixture comprises polymer precursor compounds (aka polymer building blocks) of biobased MVL or MVL derivatives, such as described in co-pending U.S. Ser. No. 14/939,437. In one or more embodiments, a polymer precursor compound of the invention comprises ring-opened biobased MVL or derivatives thereof. Examples of polymer precursor compounds of the invention include biobased MVL-diols, biobased MVL-diacids, and biobased MVL-glycidyl ether/esters. The invention facilitates the synthesis of compositions comprising one or more of the ring-opened biobased MVL compounds, including polymers prepared from one or more of the foregoing biobased MVL precursor compounds. The invention further provides a variety of other biobased polymers and oligomers prepared from biobased MVL or derivatives as described herein.

In more detail, described herein are compounds (e.g., monomers, oligomers, and/or polymers) derived from biobased compounds, and specifically biobased MVL and its related derivatives, and processes for synthesizing these compounds. Through oxidation, for example, these biobased precursors can be reacted to yield building blocks for (unsaturated-) polyesters, polyester polyols and polyamides, as well as precursors for glycidyl esters and omega-alkenyl esters (e.g., allyl ethers, homoallyl ethers, vinyl ethers, etc.).

The process steps of the invention are preferably carried out in solution, e.g., in the presence of a solvent. In some embodiments the solvent functions as a co-reactant by reacting with MVL, a derivative of MVL, or some other compound derived from MVL. In some embodiments of the invention, water is used as the solvent. Other solvents, such as linear, branched, or cyclic alcohols or diols (i.e., methanol, ethanol, propanol, isopropanol, sec-butanol, cyclobutanol, glycerol, 2-ethyl hexanol, propanediol, butanediol, lauryl alcohol, etc.) may also be used. Other suitable solvents include polar, aprotic solvents such as tetrahydrofuran (THF), other linear or cyclic ethers, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dioxane, etc. as a general matter, alkyl alcohol, ethers, aromatic compounds, acids, aldehydes, esters all can function as effective solvents and co-reactants in various embodiments of the invention, as will be readily discernible to one of skill in the art. Mixtures of water and these solvents (and other suitable solvents) may also be used.

In some embodiments of the invention, one or more of the process steps occurs in the presence of a catalyst. The catalyst can be a currently known catalyst, or a catalyst developed in the future. In some embodiments, the preferred catalyst, particularly, but not exclusively, with regard to the first step is a solid catalyst. Preferred catalysts of the invention include solid acid catalysts, metal oxide catalysts and carbon catalysts. A solid acid catalyst may comprise one or more solid acid materials without limitation, whether now known or developed in the future. Exemplary solid acid catalysts which can be utilized include, but are not limited to, heteropoly acids, acid resin-type catalysts, meso-porous silicas, acid clays, sulfated zirconia, molecular sieve materials, zeolites, and acidic material on a thermo-stable support. Where an acidic material is provided on a thermo-stable support, the thermo-stable support can include for example, one or more of silica, tin oxide, niobia, zirconia, titania, carbon, alpha-alumina, and the like. The oxides themselves (e.g., ZrO₂, SnO₂, TiO₂, etc.), which may optionally be doped with additional acid groups such as sulfates, phosphates, etc., may also be used as solid acid catalysts. Further examples of suitable solid acid catalysts include strongly acidic ion exchangers such as cross-linked polystyrene containing sulfonic acid groups. For example, the Amberlyst resins® are functionalized styrene-divinylbenzene copolymers with different surface properties and porosities. The functional group is generally that of the sulfuric acid type. The Amberlyst resins are supplied as gellular or macro-reticular spherical beads (Amberlyst® is a registered trademark of the Dow Chemical Co., Midland, Mich.). Similarly, Nafion resins® are sulfonated tetrafluoroethylene-based fluoropolymer copolymers which are solid acid catalysts (Nafion® is a registered trademark of E.I. du Pont de Nemours & Co., Wilmington, Del.). Zeolites may also be used as solid acid catalysts. Of these, H-type zeolites are generally preferred, for example zeolites in the mordenite group or fine-pored zeolites, such as zeolites X, Y, and L (i.e., mordenite, erionite, chabazite, or faujasite). Additional zeolites are the ultrastable zeolites in the faujasite group which have been dealuminated. The solid acid catalyst is preferably selected from the group consisting of functionalized styrene-divinylbenzene copolymers, functionalized tetrafluoroethylene-fluoropolymer copolymers, calcium apatite, silica-alumina, silica, titania, alumina, resins, metal oxides, and zeolites.

In some preferred embodiments of the invention, the catalysts used is selected from the group consisting of silicon oxide, aluminum oxide, titanium oxide, magnesium oxide, zirconium oxide, cobalt oxide, iron oxide, nickel oxide, manganese oxide, zinc oxide, molybdenum oxide, tungsten oxide, calcium oxide, cerium oxide, tin oxide, and chromium oxide, and mixtures thereof. Some preferred embodiments of the invention employ a solid catalysts that comprises a mixed metal oxide catalyst, i.e., a catalyst comprising two or more distinct metals or metal oxides. In some preferred embodiments, a process of the invention employs a heterogeneous catalyst. In some embodiments, potassium bisulfate is excluded from the list of solid acid catalysts, for the first catalyst.

In some embodiments, a solid catalyst used in the invention comprises a supported transition or noble metal. An illustrative, non-limiting list of transition or noble metals encompassed by the invention includes palladium, magnesium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, zirconium, molybdenum, ruthenium, rhodium, silver, tin, tungsten, tantalum, iridium, platinum, and gold, and mixtures thereof.

In some embodiments of the invention, where the second step involves reaction with an olefin, a catalyst is employed that is selected from the group consisting of ruthenium, molybdenum, tungsten, rhenium, tin, and rhodium olefin metathesis catalysts, and mixtures thereof. In other embodiments, where the second step involves reaction with an alcohol, a catalyst is employed that is selected from the group consisting of homogenous and heterogeneous acids and bases, sugars, lipases, ion exchange resins, metal oxides, and zeolites, and mixtures thereof.

Catalyst of the invention are characterized by a wide range of surface areas. In some embodiments, a solid catalyst is used having a surface area equal to or greater than 1 m²/g, 5 m²/g, 10 m²/g, 20 m²/g, 50 m²/g, 100 m²/g, or 200 m²/g. In some embodiments, a solid catalyst is used that has a surface area equal to or less than 50 m²/g, 100 m²/g, 200 m²/g, 600 m²/g, 1000 m²/g, or 2000 m²/g. As one illustrative but non-limiting example, in some preferred embodiments the solid catalyst has a surface area between 20 m²/g and 600 m²/g.

In some embodiments, the first product or first product mixture comprises one or more of isoprene, 3-methyl-2-butanone, methyl vinyl ketone, anhydro-mevalonolactone, 2-pentene, 2-methyl-1-butene, 2-methyl-1-pentene, methane, hydrogen, or an alkyl alcohol.

In some embodiments, the first product or first product mixture comprises compounds independently selected from the group consisting of

wherein R₁, R₂, R₃, R₄, and R₅ are independently selected from the group consisting of H, branched and unbranched C₁-C₃ alkyl and branched and unbranched C₂-C₄ alkenyl.

In a preferred embodiment, the first product or first product mixture comprises compounds independently selected from the group consisting of

In one embodiment, the olefin of the optional second step is preferably selected from the group consisting of branched and unbranched C₂-C₂₀ alkenes, branched and unbranched C₃-C₂₀ alkenic acids, branched and unbranched C₃-C₂₀ alkenoic acid esters, wherein the olefin is optionally substituted. The olefin is more preferably selected from the group consisting of ethene, propene, 1-butene, 2-butene, 2-methyl-2-propene, acrylic acid, palmitoleic acid, oleic acid, linoleic acid, and arachidonic acid, and esters thereof.

The second step may further comprise a second catalyst. In one embodiment the second catalyst is preferably an olefin metathesis catalyst selected from the group consisting of palladium, platinum, ruthenium, molybdenum, tungsten, rhenium, tin, and rhodium olefin metathesis catalysts, and mixtures thereof. Examples of specific catalysts can be found in the primary literature, the patent literature, and reviews, such as Grubbs, R. H. et al. J. Am. Chem. Soc. 2011, 133, 7490-7496; Kadyrov, R. et al. Top. Catal. 2012, 55, 538-542; Hoveyda, A. H. et al. J. Am. Chem. Soc. 2009, 131(31), 10840-10841; Grubbs, R. H. et al. J. Am. Chem. Soc. 2012, 134(1), 693-699; Schrodi, Y. et al. Clean 2008, 36(9), 669-673; Elevance Renewable Sciences Inc. U.S. Patent Application Publication No. 2013/0289327; and Burk, M. J. et al. U.S. Patent Application Publication No. 2009/0155866. Preferred catalysts are shown below (Grubbs, R. H. et al. J. Am. Chem. Soc. 2011, 133, 7490-7496).

In one embodiment, the alcohol of the second step is preferably selected from the group consisting of branched and unbranched C₁-C₃₄ alkyl alcohols, branched and unbranched C₂-C₂₀ alkyl diols, and branched and unbranched C₂-C₃₄ alkenols. The alcohol is more preferably selected from the group consisting of methanol, ethanol, glycerol, 2-ethyl hexanol, propanediol, butanediol, and lauryl alcohol.

The second step may further comprise a second catalyst. In one embodiment, the second catalyst is preferably selected from the group consisting of homogenous and heterogeneous acids and bases, sugars, lipases, ion exchange resins, metal oxides, and zeolites. The second catalyst is more preferably selected from the group consisting of KOH, KOCH₃, NaOCH₃, NaOH, H₂SO₄, and mixtures thereof.

The International Union of Pure and Applied Chemistry (IUPAC) names for some of the chemical compounds described herein are as follows: Mevalonic acid: 3,5-dihydroxy-3-methylpentanoic acid; Mevalonolactone: 4-hydroxy-4-methyltetrahydro-2H-pyran-2-one; Dehydrated Mevalonolactone: 4-methyl-5,6-dihydro-2H-pyran-2-one (isomer one) and 4-methyl-3,6-dihydro-2H-pyran-2-one (isomer two); Dehydrated Mevalonic acid: (E)-5-hydroxy-3-methylpent-2-enoic acid (isomer one) and (E)-5-hydroxy-3-methylpent-3-enoic acid (isomer two).

It is preferred that after the first reaction, and before the second reaction, at least a fraction of any water present in the solution containing the compound or mixture of compounds is removed. The water separation step is optional, particularly if a water-tolerant catalyst is used in the second step, or water-dilution is not undesirable.

Reactions of the invention are preferably conducted in a range from about 250K to about 1000K. In some embodiments of the invention, a reaction step is performed at a temperature equal to or greater than about 25° C., 50° C., 100° C., 150° C., 200° C., 300° C., 400° C., or 500° C. In some embodiments of the invention, a reaction step is performed at a temperature less than or equal to about 100° C., 150° C., 200° C., 300° C., 400° C., 500° C., 600° C., or 750° C. Non-limiting but illustrative examples of temperature ranges employed in certain preferred embodiments of the invention include about 100° C. to 500° C., about 150° C. to 500° C., about 200° C. to 500° C., about 250° C. to 500° C., about 100° C. to 400° C., about 150° C. to 400° C., about 200° C. to 400° C., and about 250° C. to 500° C. Reactions of the invention can be performed either in the presence or absence of a catalyst, and is a general matter lower temperatures can be employed to good effect in the presence of the appropriate catalysts, which can be readily determined by one of skill in the art.

Reactions of the invention can also be performed at a range of pressures varying from between sub-atmospheric to extremely high. In some embodiments of the invention, a reaction step is performed at a pressure greater than equal to about atmospheric pressure, 2 bar, 5 bar, 10 bar, 20 bar, 35 bar, 50 bar, 100 bar, 200 bar, 500 bar, or even higher. In some embodiments of the invention, a reaction step is performed at a pressure less than or equal to atmospheric pressure, 2 bar, 5 bar, 10 bar, 20 bar, 35 bar, 50 bar, 100 bar, 200 bar, 500 bar, or 1000 bar. Non-limiting but illustrative examples of pressure ranges employed in certain preferred embodiments of the invention include 0 to 15 bar, 2 bar to 100 bar, 5 bar to 100 bar, 10 bar to 100 bar, 20 bar to 100 bar, 35 bar to 100 bar, and 50 bar to 100 bar. In some instances the desired output of the invention can be enhanced by means of using a combination of relatively high temperature and pressure, in the presence or absence of a catalyst. For example, in some embodiments of the invention one or more of the reactions occur at a temperature in the range of 100° C. to 500° C. and a pressure in the range of 20 bar to 100 bar, or at a temperature in the range of 200° C. to 500° C. and a pressure in the range of 20 bar to 100 bar.

In one embodiment, the product or product mixture yielded from the second step are independently selected from the group consisting of

wherein R₆ is independently selected from the group consisting of branched and unbranched C₁-C₃₄ alkanes, branched and unbranched C₂-C₂₀ alkyl alcohols, and branched and unbranched C₂-C₃₄ alkenes, and more preferably selected from the group consisting of methyl, ethyl, glyceryl, 2-ethyl hexyl, 2-propanolyl, butanolyl, and lauryl.

In another embodiment, a product or product mixture of the second step may be combined at a third temperature and a third pressure with a reagent and a catalyst. In a preferred embodiment, the reagent is an olefin and the catalyst is preferably an olefin metathesis catalyst selected from the group consisting of palladium, platinum, ruthenium, molybdenum, tungsten, rhenium, tin, and rhodium olefin metathesis catalysts, and mixtures thereof. Examples of specific catalysts can be found in the primary literature, the patent literature, and reviews, such as Grubbs, R. H. et al. J. Am. Chem. Soc. 2011, 133, 7490-7496; Kadyrov, R. et al. Top. Catal. 2012, 55, 538-542; Hoveyda, A. H. et al. J. Am. Chem. Soc. 2009, /3/(31), 10840-10841; Grubbs, R. H. et al. J. Am. Chem. Soc. 2012, 134(1), 693-699; Schrodi, Y. et al. Clean 2008, 36(9), 669-673; Elevance Renewable Sciences Inc. U.S. Patent Application Publication No. 2013/0289327; and Burk, M. J. et al. U.S. Patent Application Publication No. 2009/0155866. Preferred catalysts are shown in Scheme 1 (Grubbs, R. H. et al. J. Am. Chem. Soc. 2011, 133, 7490-7496). The olefin of the third step is preferably selected from the group consisting of branched and unbranched C₂-C₂₀ alkenes, branched and unbranched C₃-C₂₀ alkenic acids, branched and unbranched C₃-C₂₀ alkenoic acid esters, wherein the olefin is optionally substituted. The olefin is more preferably selected from the group consisting of ethene, propene, 1-butene, 2-butene, 2-methyl-2-propene, acrylic acid, palmitoleic acid, oleic acid, linoleic acid, and arachidonic acid, and esters thereof.

In another preferred embodiment, the reagent is an alcohol, and the catalyst is preferably selected from the group consisting of homogenous and heterogeneous acids and bases, sugars, lipases, ion exchange resins, metal oxides, and zeolites. The catalyst is more preferably selected from the group consisting of KOH, KOCH₃, NaOCH₃, NaOH, H₂SO₄, HCl, Amberlite resin®, Amberlyst resin®, CaO—CeO₂, ZSM-5 zeolites, and mixtures thereof. The alcohol of the third step is preferably selected from the group consisting of branched and unbranched C₁-C₃₄ alkyl alcohols, branched and unbranched C₂-C₀ alkyl diols, and/or branched and unbranched C₂-C₃₄ alkenols. The alcohol is more preferably selected from the group consisting of methanol, ethanol, glycerol, 2-ethyl hexanol, propanediol, butanediol, and/or lauryl alcohol.

In another embodiment, the product or product mixture resulting from the first step comprises molecules having C₈ or less. Depending on the pH, the components may be in salt form. In some embodiments, depending on the reagent chosen, the counter ion is independently selected from the cations of ammonium, sodium, lithium, potassium, magnesium, calcium, aluminum, and/or cesium.

In some embodiments the product or product mixture comprises a compound independently selected from the group consisting of

wherein R₁, R₂, R₃, R₄, and Rs are independently selected from the group consisting of H, branched and unbranched C₁-C₃ alkyls, and branched and unbranched C₂-C₄ alkenyls.

One useful method for bio-production of a chemical product, such as biobased MVL, is fermentation. Fermentation procedures are well known to those of ordinary skill in the art. Fermentation of a set of complementary metabolizing organisms for the biosynthetic production of a target chemical compound, such as mevalonate, can be utilized in, for example, batch fermentation, fed-batch fermentation, or continuous fermentation. In addition, any of these methods of fermentation also can be coupled with well-known separation methods applicable to fermentation procedures such as batch separation or continuous separation. Exemplary combinations of fermentation and separation methods applicable for the bio-production of a target chemical compound such as mevalonate include, for example, batch fermentation and batch separation, batch fermentation and continuous separation, fed-batch fermentation and batch separation, fed-batch fermentation and continuous separation, continuous fermentation and batch separation, or continuous fermentation and continuous separation. Examples of batch and continuous fermentation procedures are well known in the art.

Mevalonate can be produced from both naturally and non-naturally occurring microbes. In some embodiments, the invention provides engineered, non-naturally occurring microbes capable of expressing MVL and/or MVL derivatives by means of a biosynthetic MVL pathway. The invention further provides processes of using both naturally and non-naturally occurring microbes capable of expressing, and preferably actually expressing, a biosynthetic MVL pathway in the production of biobased MVL and MVL derivatives. For example, in some embodiments the invention provides a process that comprise steps of (a) providing a microbial organism that expresses a biosynthetic MVL pathway; and (b) growing the microbial organism in fermentation medium comprising suitable carbon substrates, whereby biobased MVL is produced.

In some preferred embodiments, of the invention, invention provides a process that comprise steps of (a) providing a microbial organism that expresses a biosynthetic MVL pathway; (b) growing the microbial organism in fermentation medium comprising suitable carbon substrates, whereby biobased MVL is produced; and (c) reacting said biobased MVL to yield a product or product mixture. In some embodiments, the reaction occurs in the presence of a catalyst, preferably a solid catalyst. In some embodiments, the reaction occurs at elevated temperature and/or pressure. In some embodiments, this first product or product mixture is subsequently reacted further in one or more subsequent reactions. For example, the first product or product mixture can be reacted with an alcohol or olefin, optionally in the presence of a catalyst, and optionally at elevated temperature and/or pressure, as described above.

In some embodiments the invention provides methods for creating non-naturally occurring microbial organism by introducing a metabolic modification, for example a metabolic modification that enables biobased MVL production. In some preferred embodiments, the metabolic modification includes at least one of the following gene sets: i. mvaE, mvaS; ii. mvaE, mvaS, atoB; and/or iii. atoB, hmgR, hmgS, or a functional homolog (or ortholog) thereof. The phrase “functional homolog” refers to a polynucleotide or polypeptide sequences that are similar due to common ancestry and sequence conservation and have identical or similar function at the catalytic, cellular, or organismal levels. Such a metabolic modification can enable stable production of mevalonate. The invention further provides the resulting non-naturally occurring microbial organisms, and methods of using the same in various processes embodying varying aspects of the invention. The mevalonate produced in any of the above-discussed processes may be used in integrated processes, such that the mevalonate is not isolated prior to the next step, or separated prior to the next step, as discussed above.

In some embodiments, the metabolic modifications further comprise modification or disruption of at least one gene. In a preferred embodiment, the gene is independently selected from the group consisting of adh1-adh7, gpd1, gpd2, ackA-pta, adhE and ldhA. In one embodiment, the non-naturally occurring microbial organism is selected from the group consisting of bacterium, yeast, algae, and fungus. Exemplary bacterium include species selected from the group consisting of E. coli, A. succiniciproducens, A. succinogenes, M succiniciproducens, R. etli, Bacillus subtilis, Corynebacterium glutamicum, Gluconobacter oxydans Zymomonas mobilis, Lactococcus lactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridium acetobutylicum, Clostridium sp., Synechococcus elongates, Pseudomonas fluorescens, Methanosarcina sp., Methylococcus sp. and Pseudomonas putida. Exemplary yeast include species selected from the group consisting of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger, Rhizopus oryzae, Rhizopus arrhizus, and Pichia sp. In some embodiments the microbial organism is a methanotrophic or photosynthetic microorganism, such as a methanotrophic or photosynthetic bacteria.

In one embodiment mevalonate can be produced utilizing naturally occurring Saccharomycopsis fibuligera IFO 0107 (Koike et al. J. Ferm. Bioeng. 1989, 68(1), 58-59). In another embodiment, mevalonate (MVA) was produced in a recombinant E. coli expressing genes for MVA biosynthesis. HMG-CoA synthase (mvaS) and bi-functional HMG-CoA reductase/acetyl CoaA acetyltransferase (mvaE) and acetyl CoaA acetyltransferase (atoB) were cloned to provide a route for the production of mevalonate in E. coli utilizing the endogenous acetyl-CoA pool. A similar process is demonstrated in the literature (Zhang, K. et al. PNAS 2014, 111(23), 8357-8362; Hashimoto, S.-I. et al. Biotech. Let. 2004, 26, 1487-1491; Endo, A. et al. J. Ferm. Bioengin. 1989, 68(1), 58-59).

In another embodiment, E. coli strain BL21(DE3), overexpressing codon optimized E. feacalis mvaE and mvaS genes driven by the T7 promoter, is cloned to provide a route for the production of mevalonate.

In another embodiment, E. coli strain S. cerevisiae CEN.PK, overexpressing codon optimized E. feacalis mvaE and mvaS genes, was cloned to provide a route for the production of mevalonate.

In another embodiment, E. coli strain BL21(DE3), overexpressing codon optimized S. cerevisiae hmgS and hmgR genes along with E.coli atoB gene, all driven by the T7 promoter, was engineered to provide a route for the production of mevalonate.

In another embodiment, mevalonate can be produced using Clostridium sp. MT1243 utilizing synthesis gas or a CO₂/H₂ blend as demonstrated in the literature (Kiriukhin, M. et al., Bioprocess Biosyst. Eng. 2014, 37, 245-260). Synthesis gas or syngas is available from a variety of carbonaceous materials, such as coal, acid-hydrolyzed lignocellulosic biomass, recovery of gas from steel mill emissions, or natural gas.

In another embodiment, Chlorella protothecoides, overexpressing codon optimized E. faecalis mvaS and mvaE genes along with E.coli atoB gene, all driven by the CaMV35S promoter, is engineered to provide a route for the production of mevalonate from CO₂ either under photoautotrophic, mixotrophic, or heterotrophic conditions.

In another embodiment, Methylococcus capsulatus, overexpressing codon optimized E. faecalis mvaS and mvaE genes along with E.coli atoB gene, all driven by the Trc promoter, is engineered to provide a route for the production of mevalonate from methane or methanol.

Fermentation of microbial organisms to produce mevalonate requires a source of carbon and energy, such as glucose, sucrose, xylose, arabinose, galactose, mannose, fructose, CO, H₂, or CO₂, and light or a combination of aforementioned. Exemplary materials and/or substrates supplying these energy sources include biomass, renewable feedstocks, natural gas, biogas, coal, and crude oil. Biomass is defined as any plant-derived organic material. Optimally, biomass for energy is obtained from a sustainable basis, such as herbaceous and woody energy crops, agricultural food and feed crops, agricultural crop wastes and residues, wood wastes and residues, aquatic plants, and other waste products (i.e., some municipal wastes). Sources of biomass that can be used as feedstocks are cellulosic biomass, hemicellulosic biomass, or lignocellulosic biomass, such as agricultural residue (i.e., wheat strass, corn stover, bagasse, wheat or cotton woodchips), reed canary grass, corn, wheat, cotton, wood chips, or energy crops (i.e., miscanthus). A renewable feedstock is defined as a renewable energy source, such as material derived from living organisms or their metabolic byproducts, including material derived from biomass. In addition to utilizing agricultural residue, crops grown for use as renewable feedstock include corn, soybeans, switchgrass, wheat, flaxseed, sugarcane, palm oil, and trees (i.e., poplar).

Biomass and renewable feedstocks are particularly useful sources of a variety of carbohydrates. In particular, glucose is available from a corn dry-grind process as demonstrated in the literature (Kwiakowski, J. R. et al. Ind. Crops Prod. 2006, 23, 288-296). For an example of the corn stover conversion to glucose, see Dudgeon, D. et al., Process Design and Economics for Biochemical Conversion of Lignocellulosic Biomass to Ethanol: Dilute-Acid Pretreatment and Enzymatic Hydrolysis of Corn Stover (National Renewable Energy Laboratory (NREL), Golden, Colo., 2011).

Previously-discussed biomasses and renewable feedstocks are useful sources for a variety of carbohydrates that can be used in a growth medium for complementary metabolizing organisms for the biosynthetic production. Particularly useful carbon sources include medium or feedstocks containing different simple or complex carbohydrates, useful for cellular proliferation. In one embodiment, nutrients and media comprise at least one carbon substrate selected the group consisting glucose, sucrose, xylose, arabinose, galactose, mannose, fructose, CO, and CO₂. In a preferred embodiment, the nutrients and media are a renewable feedstock. In one embodiment, the renewable feedstock is derived from biomass. In a preferred embodiment, the renewable feedstock is selected from the group consisting of a cellulosic biomass or a hemicellulosic biomass. In another embodiment, the renewable feedstock comprises a carbon source selected from the group consisting of carbohydrates, aromatic compounds, and lignin.

Additional advantages of the various embodiments of the invention will be apparent to those skilled in the art upon review of the disclosure herein and the working and/or prophetic examples provided below. It will be appreciated that the various embodiments described herein are not necessarily mutually exclusive unless otherwise indicated herein. For example, a feature described or depicted in one embodiment may also be included in other embodiments, but is not necessarily included. Thus, the present invention encompasses a variety of combinations and/or integrations of the specific embodiments described herein.

As used herein, the phrase “and/or,” when used in a list of two or more items, means that any one of the listed items can be employed by itself or any combination of two or more of the listed items can be employed. For example, if a composition is described as containing or excluding components A, B, and/or C, the composition can contain or exclude A alone; B alone; C alone; A and B in combination; A and C in combination; B and C in combination; or A, B, and C in combination.

The present description also uses numerical ranges to quantify certain parameters relating to various embodiments of the invention. It should be understood that when numerical ranges are provided, such ranges are to be construed as providing literal support for claim limitations that only recite the lower value of the range as well as claim limitations that only recite the upper value of the range. For example, a disclosed numerical range of about 10 to about 100 provides literal support for a claim reciting “greater than about 10” (with no upper bounds) and a claim reciting “less than about 100” (with no lower bounds). Furthermore, whenever a number is provided that constitutes an upper or lower limit, or a numerical range, such as “equal or greater than 100,” “less than or equal to 100,” or “between 10 and 100,” it should be understood that such limit or numerical range is to be construed as providing literal support for claim limitations that incorporate the word “about” prior to a numerical term. For example, “between 10 and 100” should be understood to provide literal support for a claim limitation reciting “between about 10 and between about 100.”

EXAMPLES

The following examples set forth preferred methods in accordance with the invention. It is to be understood, however, that these examples are provided by way of illustration and nothing therein should be taken as a limitation upon the overall scope of the invention. In addition, all examples conducted with purified mevalonate can also be conducted with unpurified material.

Example 1 Plasmid and E.coli Strain Construction

Genetic segments encoding mvaE (acetyl-CoA acetyltransferase/HMG-CoA reductase, GenBank No. AAG02438) and mvaS (HMG-CoA synthase, GenBank No. AAG02439) in

Enterococcus faecalis V583 were amplified from its genomic DNA (obtained from ATCC). These segments were inserted into a vector (with pBR322 origin back bone, Ampicillin marker, lacIq, rrnB transcription termination sequences) under the control of IPTG inducible Trc promoter-lac operator to obtain plasmid pSEl (SEQ ID NO:1).

Chemically competent E.coli cells of XL-lBlue strain (endA1 gyrA96(nal^(R)) thi-1 recA1 relA1 lac glnV44 F[ ::Tn10 proAB⁺ lacI^(q) Δ(lacZ)M15] hsdR17(r_(K) ⁻ m_(K) ⁻)) were transformed with plasmid pSEl using the procedures outlined in Sambrook-Maniatis (Green, M. R.; Sambrook, J. Ed. Molecular Cloning: A Laboratory Manual, Fourth Edition, 2002) to obtain strain E.coli-SEl.

Example 2 Mevalonolactone Production

E.coli-SE1 strain was propagated in LB medium supplemented with 100 μg/liter ampicillin in 250 mL of media in a 1 liter conical flask incubated at 37° C. in an orbital shaker at 220 rpm for 10 hours reaching an OD600 of 3. This was used as an inoculum for the production in an Infors 51t bioreactor. 1.75 liter of production media (containing 15 g/l glucose, 7 g/lKH₂PO₄, 1 g/l NH₄Cl, 5 g/l yeast extract, 1 g/l citric acid, 10 mg MnSO₄, 2 g/l MgSO₄, 200 mg/l FeSO₄ and 10 mg/l thiamine.HCl) was combined with 250 mL of inoculum in the bioreactor. The pH was maintained at 7 with 20% NH₄OH. Temperature was maintained at 32° C. Air was sparged at 2 liters per minute (LPM) and agitation was maintained at 700 rpm. 10 hours after inoculation, 1 ml of 1M IPTG was added to the bioreactor. Anti-foam was added as needed. Glucose concentration was maintained around 10 g/l, by addition of 600 g/l glucose solution to the bioreactor at 2 hour intervals. The bioreactor run was stopped at 48 hours. Cells were separated from the broth by use of 0.45 micro filters to obtain clear broth. Mevalonolactone concentration was found to be 40 g/l at end of the fermentation.

Example 3 Mevalonolactone Purification

The clear broth from Example 2 was concentrated by evaporation in a rotary evaporator to a volume of 400 mL, and acidified to pH 2 by addition of 20% H₂SO₄. NaCl was added until the solution was saturated with it. Mevalonolactone was extracted into ethyl acetate by using 200 mL four times to obtain 800 mL of ethyl acetate. This was concentrated to 200 mL in a rotary evaporator. Mevalonolactone was back extracted into 150 ml 10M NaOH solution from the ethyl acetate. The acidification and ethyl extraction steps were repeated and all the ethyl acetate was evaporated to obtain mevalonolactone with purity over 95%.

Example 4 Plasmid and Cyanobacterial Strain Construction

Genetic segments encoding mvaE (acetyl-CoA acetyltransferase/HMG-CoA reductase, GenBank No. AAG02438) and mvaS (HMG-CoA synthase, GenBank No. AAG02439) in Enterococcus faecalis V583 are amplified from its genomic DNA (obtained from ATCC). These segments are inserted into vector MCS under the control of constitutive promoter, psbA1, to obtain plasmid pMSEl.

The construct is inserted into the Cyanobacterium Synechococcus elongatus strain PCC 7942 genome using plasmid pMSE1 as per manufacturer's procedures (Invitrogen, GeneArt® Synechococcus Protein Expression Kit, Publication No. MAN0009792, pages 16-17) to obtain strain S. elongatus-MSE1.

Example 5 Photosynthetic Production of Mevalonolactone

S. elongatus-MSE1 strain is propagated in BG-11 medium supplemented with 50 mM NaHCO₃ and 10 mg/l thiamine. Cells are grown in 600 mL of media in a 1 liter Roux bottle incubated at 30° C. under fluorescent light (55 μE s⁻¹ m⁻²), which is provided by eight 86-cm 20-W fluorescent tubes placed 15 cm from the cell culture and aerated by air containing 5% CO₂. Daily, one-tenth the total volume of cell culture is removed from the cell culture. Then the same volume of fresh medium containing 0.5 M NaHCO₃ is added to cell culture. The pH of the cell culture with NaHCO₃ is adjusted to 7.5 with 10 N HCl every day. This is done for 10 days.

Example 6 Production of Mevalonolactone from Acetate

E.coli-SE1 strain was propagated in LB medium supplemented with 100 μg/liter ampicillin in 5 mL, of media in a 15 ml tube incubated at 37° C. in an orbital shaker at 220 rpm for 10 hours reaching an OD600 of 3. This was used as an inoculum for the production in a 250 ml shake flask. 50 mL of production media (containing 5 g/l sodium acetate, 7 g/l KH₂PO₄, 1 g/l NH₄Cl, 5 g/l yeast extract, 1 g/l citric acid, 10 mg MnSO₄, 2 g/l MgSO₄, 200 mg/l FeSO₄ and 10 mg/l thiamine.HCl) was combined with 5 mL of inoculum in the shake flask. The pH was maintained at 7 with 20% NH₄OH. Temperature was maintained at 32° C. 10 hours after inoculation, 25 g/l of 1M IPTG was added to the flask. Acetate concentration was maintained around 2 g/l by addition of 300 g/l sodium acetate solution to the shake flask at 6 hour intervals. The run was stopped at 72 hours. Cells were separated from the broth by use of 0.45 micro filters to obtain clear broth. Mevalonolactone concentration was found to be 8 g/l at end of the fermentation.

Example 7 Production of Mevalonolactone from Glycerol

E.coli-SE1 strain was propagated in LB medium supplemented with 100 μg/liter ampicillin in 5 mL, of media in a 15 ml tube incubated at 37° C. in an orbital shaker at 220 rpm for 10 hours reaching an OD600 of 3. This was used as an inoculum for the production in a 250 ml shake flask. 50 mL, of production media (containing 10 g/l glycerol, 7 g/l KH₂PO₄, 1 g/l NH₄Cl, 5 g/l yeast extract, 1 g/l citric acid, 10 mg MnSO₄, 2 g/l MgSO₄, 200 mg/l FeSO₄ and 10 mg/l thiamine.HCl) was combined with 5 mL of inoculum in the shake flask. The pH was maintained at 7 with 20% NH₄OH. Temperature was maintained at 32° C. 10 hours after inoculation, 25 g/l of 1M IPTG was added to the flask. Glycerol concentration was maintained around 5 g/l by addition of 400 g/l aqueous glycerol solution to the shake flask at 6 hour intervals. The run was stopped at 72 hours. Cells were separated from the broth by use of 0.45 micro filters to obtain clear broth. Mevalonolactone concentration was found to be 15 g/l at end of the fermentation.

Example 8 Production of Mevalonolactone from Syn-Gas

Production of mevalonate from syn-gas (mixture of CO₂, CO and H₂ in various ratios) is described by Kiriukhin et al. (“Mevalonate production by engineered acetogen biocatalyst during continuous fermentation of syngas or CO₂/H₂ blend,” Bioprocess Biosyst. Eng. 2014 37, 245-260), and is herein incorporated in its entirety.

Example 9 Plasmid and Yeast Strain Construction

The promoter and terminator DNA sequences were synthesized by Biobasic, Inc, Canada. Codon optimized gene sequences for the enzymes mvaE and mvaS-(A110G) from E. faecalis were synthesized by Biobasic. Sequences encoding mvaE under control of TDH3 promoter, and ADH1 terminator and mvaS-(A110G) under control of TEF1 promoter and ACT1 terminator, were cloned into a vector (which contains a 2 micron origin, URA3 marker and Ampicillin marker) to obtain plasmid pVSl9 (SEQ ID NO:2).

Yeast S.cerevisiae CEN.PK2-1C (MATa; ura3-52; trp1-289; leu2-3,112; his3Δ. 1; MAL2-8^(c); SUC2) was transformed with plasmid pVSl9 using the procedures outlined in Sambrook-Maniatis (Green, M. R.; Sambrook, J. Ed. Molecular Cloning: A Laboratory Manual, Fourth Edition, 2002) to obtain stain S.cerevisiae-VS19.

Exampl 10 Production of Mevalonolactone from Sucrose

S.cerevisiae-VS19 strain was propagated in 5 ml of CM Glucose Broth minus Uracil (Teknova catalogue no. C8140) media in a 15 ml tube incubated at 30° C. in an orbital shaker at 220 rpm for 72 hours. Cells were separated from the broth by use of 0.45 micro filters to obtain clear broth. Mevalonolactone concentration was found to be 1.2 g/l at end of the fermentation.

Example 11 Initial Reactor Conditions Fixed Bed Reactor Set-up

Reactions were performed in a high pressure small scale test unit (FIG. 1) equipped with three gas lines, controlled by high accuracy mass flow controllers and one liquid feed line, delivering liquid feedstocks via a high precision pump. The unit operates with a stainless steel fixed bed reactor, externally heated with a three-zone furnace, while the exit stream of the reactor is cooled via a heat exchanger and is directed to a system of vessels for the separation and collection of the liquid and gaseous products. The reaction temperature was monitored with a thermocouple inserted in the catalytic bed. Both the liquid products and the gaseous stream were analyzed with gas chromatography.

Catalyst & Feedstock

Tests were conducted with a commercial amorphous SiO₂/Al₂O₃ catalyst supplied by Grace (DAVICAT SIAL 3113) and a commercial ZSM-5 (SiO₂/Al₂O₃ ratio 23-CBV2314) supplied by Zeolyst Inc. In both cases, the catalysts were supplied in powder form. The amorphous SiO₂/Al₂O₃ was used as provided. The ZSM-5 catalyst was calcined in air at 500° C. for 3 h in order to convert from the ammonium to the H^(i)-form. The sample was then crushed and sieved to a particle size of 100-180 μm prior to use. The tests were performed with 10 wt % and 20 wt % aqueous solutions of mevalonolactone, and 10 wt % dehydro-mevalonolactone (also referred to as anhydro-mevalonolactone).

Catalyst Loading

A plug of glass wool was first inserted in the reactor and after packing, the reactor was filled with the necessary catalyst amount. On top another plug of glass wool was inserted, and the reactor was then connected to the unit.

Experimental Procedure and Conditions

The mevalonolactone conversion tests were performed in the down-flow stainless steel fixed bed reactor loaded with the appropriate amount (14.3 g) of catalyst. The catalyst was pretreated in-situ at 300° C. in air for 30 min. Prior to feed introduction, the desired reaction temperature and pressure was achieved under flowing inert gas (N₂). Upon reaching the desired reaction conditions, the aqueous solution of mevalonolactone (10 wt % mevalonolactone) was fed to the packed tubular reactor using a high precision pump. In order to maintain the pressure, a small N₂ flow (50 cm³/min) was co-fed with the liquid feed. The reaction was conducted at a pressure of 36 bar using a weight hourly space velocity (WHSV) of 1 h⁻¹or 2 h⁻¹. The WHSV was calculated based on the total liquid feed (solution). Steady-state activity measurements were taken after ˜2 hours time on-stream for each condition. Liquids were collected in a trap (˜10° C.), while gaseous samples were collected in gas sampling bags.

Product Analysis

Analysis of gaseous products was performed on a GC equipped with two detectors (FID and TCD) and three columns (MS, Porapak N and Al₂O₃/KCl) in a series-bypass configuration. The liquids were analyzed on GC-MS.

TABLE 1 Reaction conditions for Example 11 T Pressure WHSV Condition Catalyst [° C.] [bar] [h⁻¹] Feed 1 Amorphous 200 36 1 10% aq solution SiO₂/Al₂O₃ mevalonolactone 2 Amorphous 300 36 1 10% aq solution SiO₂/Al₂O₃ mevalonolactone 3 Amorphous 400 36 1 10% aq solution SiO₂/Al₂O₃ mevalonolactone 4 ZSM-5 200 36 1 10% aq solution mevalonolactone 5 ZSM-5 400 36 1 10% aq solution mevalonolactone

The analysis of the liquid products of the reaction utilizing amorphous SiO₂/Al₂O₃ with GC-MS showed no presence of the feed (i.e., mevalonolactone) in the product in any of the investigated temperatures, indicating that full conversion of mevalonolactone was achieved in the conditions studied. The composition of the liquid products by GC-MS and the gaseous products as a function of temperature is presented in Tables 2 and 3 respectively.

TABLE 2 Analysis of the liquid products by GC-MS obtained over amorphous SiO₂/Al₂O₃ Conditions 1 2 3 WHSV, h⁻¹ 1 1 1 Pressure, bar 36 36 36 Temperature, ° C. 200 300 400 GC-MS analysis, area % Lower Upper Mevalonolactone Dehydromevalonic lactone 93.4 20.99 2-methyl-2-Propanol 1.4 2.67 2-methyl-1,3-Butadiene 1.1 4 2-methyl-3-Buten-2-ol 1.06 3.2 3-methyl-2-Butanone 1.465 45.74 2.8 88.945 3-methyl-3-Buten-1-ol 10.23 2-hydroxy-Butanoic acid 5.48 2-Butanone 3.365 Acetic acid 1.07 2.74 Propylene Glycol 1.68 Ethyl alcohol 1.59 1,3-Pentadiene 1.7 2,2-dimethyl-Propanal 1.19 1-(methylethoxy)-2-Propanol 1.95 1-methyl-2-(1-methylethyl)- 1.67 3.15 Benzene 1,2,4,5-tetramethyl-Benzene 1.54 6.85 1-methyl-4-(1-methylethyl)- 6.06 Benzene Benzene, 4-ethyl-1,2-dimethyl- 3.92 Benzene, pentamethyl- 3.67 Benzene, 1,2,3,4-tetramethyl- 3.63 Benzene, 1,2,3-trimethyl- 3.48 Benzene, 1,3-dimethyl-5-(1- 2.72 methylethyl)- Benzene, 2-ethyl-1,4-dimethyl- 2.71 Butanal, 2-methyl- 2.06 Benzene, pentamethyl- 1.7 Benzene, 4-ethyl-1,2-dimethyl- 1.65 Benzene, (1-methylethyl)- 1.61 .alpha., .beta., .beta.- 1.5 Trimethylstyrene Other* 48.49 Total 96.625 94.64 100 100

The main product obtained in the liquid layer was anhydro-mevalonolactone. Thus, at low temperature only dehydration reactions occur. Limited amounts of CO₂, along with some C₆ hydrocarbons, were detected in the gaseous products.

As the temperature was increased to 300° C., the liquid product separated into two distinct phases: an oily phase, forming a top layer, and an aqueous phase which resembled an emulsion and accumulated on the bottom. The two phases were separated and analyzed separately. The aqueous phase consisted mainly of 3-methyl-2-butanone, 3-methyl-3-buten-1-ol, and anhydro-mevalonolactone, demonstrating that both dehydration and ring opening reactions were taking place. The oily phase was very complex and consisted of over 150 compounds in small concentrations. The analysis shown in Table 2 represents the components detected with a concentration higher than 1.5%. The most abundant compounds in this phase were aromatic hydrocarbons with a benzene ring. A small amount of isoprene was detected in both the oily and the aqueous phase at 300° C. The gaseous product (Table 3) showed increased CO₂ production (higher extent of severe cracking reactions) and small amounts of light hydrocarbons.

When the temperature was raised to 400° C., the liquid product turned back to a homogeneous one phase product, with an emulsion-like texture. In this case, no anhydro-mevalonolactone was detected, and the major product was 3-methyl-2-butanone. A significant amount of light alkanes and olefins, ranging from Ci to Cs were also produced, indicating that at such high temperature (400° C.), extensive decarboxylation and cracking of the lactone feed occurred.

TABLE 3 Analysis of the gaseous products obtained over amorphous SiO₂/Al₂O₃ Conditions 1 2 3 WHSV, h⁻¹ 1 1 1 Pressure, bar 36 36 36 Temperature, ° C. 200 300 400 GC analysis vol % Methane 0.234 Ethane 0.045 Ethylene 0.043 Propane 0.04 Propylene 0.024 0.22 Isobutane 0.106 n-butane 0.009 1-butene 0.009 0.071 Isobutylene 0.035 Cis-2-butene 0.281 0.75 1.212 Isopentane 0.005 0.037 n-pentane 0.038 C₅₊ 0.01 0.142 1.037 C₆₊ 1.894 1.621 0.183 CO₂ 4.354 10.916 9.744 CO 0.124 0.317 N₂ 95.613 88.865 91.398 Total 102.209 102.495 104.896 Catalytic Results with ZSM-5

As in the case of amorphous SiO₂/Al₂O₃, unconverted mevalonolactone was not detected in the liquid product of any of the investigated temperatures with ZSM-5 catalysis. This indicated that full conversion of mevalonolactone was also achieved under the conditions studied with ZSM-5. The composition of the liquid products by GC-MS and the gaseous products as a function of temperature over ZSM-5 is presented in Tables 4 and 5 respectively.

At 200° C., the liquid product of the reaction was an emulsion-like homogeneous solution. The main products obtained are the anhydrous form of mevalonolactone and 3-methyl-2-butanone. Limited amounts of CO₂ were detected in the gas products. Compared to the amorphous silica-alumina, where only dehydrated mevalonolactone was observed at 200° C., the much more acidic ZSM-5 catalyst not only catalyzed dehydration, but also decarboxylation.

At 400° C., the liquid product separated into two distinct phases: an oily phase, forming a top layer, and an aqueous emulsion-like phase. The two phases were again separated and analyzed separately. The aqueous phase consisted mainly of acetic/propanoic acid, acetone, and toluene. In contrast to 200° C. and the results with amorphous silica alumina, no 3-methyl-2-butanone was observed. This indicates the occurrence of extensive decarboxylation/cracking reactions. The oily phase was comprised of aromatic compounds, such as p-xylene, toluene, 1,2,3-trimethyl-benzene, and 1-ethyl-2-methyl-benzene. These aromatics are probably a result of oligomerization reactions of olefins which form as intermediates over ZSM-5. CO₂ and small amounts of light hydrocarbons (alkanes/alkenes) were detected in the gas phase, as shown in Table 5.

TABLE 4 Analysis of liquid products by GC-MS obtained over ZSM-5 Conditions 4 5 WHSV, h⁻¹ 1 1 Pressure, bar 36 36 Temperature, ° C. 200 400 GC-MS analysis, area % Lower Upper Mevalonolactone Dehydromevalonic lactone 66.15 No matches found 0.31 6.04 Ethyl alcohol 0.28 3.10 Butane 0.55 2-Propanol, 2-methyl- 3.28 Propanal, 2,2-dimethyl- 0.56 2-Butanone 0.32 Butanal, 2,2-dimethyl- 0.3 2-Butanone, 3-methyl- 15.75 Acetone 23.10 2-Butanone 5.44 Acetic acid 0.35 40.50 Propanoic acid 5.07 Isopropyl Alcohol 4.62 2-Propanol, 1-(1-methylethoxy)- 5.10 p-Xylene 3.79 22.7 o-xylene 1.11 Benzene, 1,2,3-trimethyl- 13.34 Benzene, 1-ethyl-2-methyl- 1.96 12.58 Toluene 1.96 7.03 11.39 Benzene, 1,3-dimethyl- 6.92 Benzene, 4-ethyl-1,2-dimethyl- 3.45 2-Tolyloxirane 1.42 Benzene, 1,3,5-trimethyl- 2.59 Benzene, 1-methyl-2-(1- 0.73 methylethyl Benzene, 1-methyl-3-propyl- 1.21 Naphthalene, 1-methyl- 1.16 Naphthalene, 2,7-dimethyl- 1.07 1-Phenyl-1-butene 1 Benzene, (1-methyl-1-buteny1)- 0.89 Benzene, 1-methyl-4-(1- 0.86 methylethyl Benzene, 1,3-diethyl- 0.86 Benzene, 2-butenyl- 0.69 Benzene, propyl- 0.6 Naphthalene, 1,2,3,4-tetrahydro-6- 1.54 methyl- Other* 19.32 TOTAL 100 100 100

TABLE 5 Analysis of gaseous products obtained over ZSM-5 Conditions 4 5 WHSV, h⁻¹ 1 1 Pressure, bar 36 36 Temperature, ° C. 200 400 GC analysis, vol % Hydrogen 0.06 Methane Ethane Ethylene Propane 0.46 Propylene 0.06 Isobutane 0.02 0.68 n-butane 0.14 1-butene Isobutylene 1.15 0.05 Trans-2-butene 0.02 Cis-2-butene 1,3-butadiene Isopentane 0.22 n-pentane 1-pentylene C₅₊/C₆₊ 0.55 0.34 CO₂ 8.58 4.65 (??) CO 0.47 N₂ 93.26 90.20 Total 103.55 97.35

Example 12 Effect of Temperature over Amorphous SiO₂/Al₂O₃

The reactions in Example 12 were run in an analogous procedure to that described in Example 11.

TABLE 6 Reaction Conditions in Examples 12 and 13 T Pressure WHSV Condition Catalyst [° C.] [bar] [h⁻¹] Feed 1 Amorphous 275 36 1 10% aq solution SiO₂/Al₂O₃ dehydro- mevalonolactone (product of reaction at 200° C. with ASA) 2 Amorphous 325 36 1 10% aq solution SiO₂/Al₂O₃ mevalonolactone 3 Amorphous 325 36 2 10% aq solution SiO₂/Al₂O₃ mevalonolactone 4 Amorphous 325 36 1 20% aq solution SiO₂/Al₂O₃ mevalonolactone

The composition of the liquid products by GC-MS and the gaseous products at 275° C. and 325° C. (conditions 1 and 2 of Table 6) are presented in Tables 7 and 8, respectively. The results for the previously investigated temperatures (200, 300 and 400° C.) are also presented for comparison reasons. The analysis of the liquid products of the reaction with GC-MS showed no presence of the feed (i.e., mevalonolactone) in the product in any of the investigated temperatures, indicating that full conversion of mevalonolactone was achieved in the conditions studied.

TABLE 7 Analysis of liquid products by GC-MS obtained over amorphous SiO₂/Al₂O₃ at experiments with constant WHSV and pressure and varying temperature WHSV, h⁻¹ 1 1 1 1 1 Pressure, bar 36 36 36 36 36 Temperature, ° C. 200 275 300 325 400 Visual inspection GC-MS analysis, area % Lower Upper Mevalonolactone Dehydromevalonic 93.4 20.99 17.55 lactone 2-methyl-2-Propanol 1.4 2.67 7.1 2-methyl-1,3- 1.1 4 5.42 Butadiene 2-methyl-3-Buten-2-ol 1.06 3.2 3.2 3-methyl-2-Butanone 1.465 78.05 45.74 2.8 44.51 88.945 3-methyl-3-Buten-1-ol 10.23 2.31 2-hydroxy-Butanoic 5.48 acid 2-Butanone 3.365 Acetic acid 1.07 2.74 Propylene Glycol 4.83 1.68 Ethyl alcohol 3.83 1.59 2.56 1,3-Pentadiene 1.7 2,2-dimethyl-Propanal 4.04 1.19 1-(methylethoxy)-2- 1.95 Propanol 1-Butanol, 4-methoxy- 3.01 Ethanol, 2-(2- 3.45 methoxyethoxy)- 1-methyl-2-(1- 1.67 3.15 methylethyl)-Benzene 1,2,4,5-tetramethyl- 1.54 6.85 Benzene 1-methyl-4-(1- 6.06 methylethyl)-Benzene Benzene, 4-ethyl-1,2- 3.92 dimethyl- Benzene, pentamethyl- 3.67 Benzene, 1,2,3,4- 3.63 tetramethyl- Benzene, 1,2,3- 3.48 trimethyl- Benzene, 1,3- 2.72 dimethyl-5-(1- methylethyl)- Benzene, 2-ethyl-1,4- 2.71 dimethyl- Butanal, 2-methyl- 2.06 Benzene, pentamethyl- 1.7 Benzene, 4-ethyl-1,2- 1.65 dimethyl- Benzene, (1- 1.61 methylethyl)- .alpha.,.beta.,.beta.- 1.5 3.45 Trimethylstyrene 1.3Cyclohexadiene, 1- methyl-4- (1methylethyl) Other 11.06 48.49 5.75 Total 96.625 99.99 94.64 100 100.13 100

TABLE 8 Analysis of gaseous products obtained over amorphous SiO₂/Al₂O₃ at experiments with constant WHSV and pressure and varying temperature WHSV, h⁻¹ 1 1 1 1 1 Pressure, bar 36 36 36 36 36 Temperature, 200 275 300 325 400 ° C. GC analysis, vol % Methane 0.234 Ethane 0.045 Ethylene 0.043 Propane 0.04 Propylene 0.006 0.024 0.007 0.22 Isobutane 0.004 0.106 n-butane 0.009 1-butene 0.009 0.004 0.071 Isobutylene 0.035 Cis-2-butene Isopentane n-pentane Cis-2-butene 0.281 0.31 0.75 0.280 1.212 Isopentane 0.005 0.003 0.037 n-pentane 0.038 C₅₊ 0.01 0.033 0.142 0.045 1.037 C₆₊ 1.894 0.811 1.621 1.260 0.183 CO₂ 4.354 2.975 10.916 3.053 9.744 CO 0.05 0.124 0.063 0.317 N₂ 95.613 97.385 88.865 97.151 91.398 Total 102.209 101.654 102.495 101.973 104.896

The main product obtained at 275° C. was 3-methyl-2-butanone, while no isoprene was formed. At 325° C., isoprene was formed in measurable amounts, together with 3-methyl-2-butanone, 2-methyl-2-propanol, and dehydro-mevalonolactone. The presence of the anhydrous form was unexpected at 325° C., as dehydration seemed to be completed even at 275° C. This inconsistency however can be explained by the fact that the feed for the experiment at 275° C. was dehydro-mevalonolactone (obtained at a previous experiment with ASA at 200° C.), while the experiment at 325° C. was conducted with a mixture of dehydro-mevalonolactone and fresh feed (i.e., mevalonolactone).

The gaseous product (Table 8) does not show a constant trend in CO₂ emissions with temperature, which can again be attributed to the different feeds used for the investigated conditions. It is clear however, that the concentration of light hydrocarbons increased with temperature, indicative of a higher extent of cracking reactions.

Since the optimum results in terms of isoprene formation were obtained at 325° C., this temperature was selected for further investigation of the effect of WHSV and feed dilution, in the decarboxylation of mevalonolactone over amorphous SiO₂/Al₂O₃.

Example 13 Effect of Feed Concentration and WHSV Over amorphous SiO₂/Al₂O₃

An analogous procedure to Example 11 was used. The reaction conditions are described in Table 6. Tables 9 and 10 present the composition of the liquid and gaseous products, respectively, obtained at a higher WHSV and with an increased mevalonolactone concentration in the feed. The reference conditions (WHSV=1 h⁻¹, 10% aq solution) are also presented in the first column for comparison reasons.

However, we see a clear increase in the production of isoprene, indicating that it is an intermediate product, and therefore favored at lower residence times. An increase was also observed in the concentration of 2-methyl-2-propanol, while the formation of 3-methyl-2-butanone seems unaffected.

Major changes were detected when the feed concentration of mevalonolactone was increased from 10 to 20 wt %. An order of magnitude increase in isoprene formation was recorded with the higher concentration feed. The only other major product with the 20 wt % mevalonolactone feed was 3-methyl-2-butanone. These results suggest that in order to increase the amount of isoprene, higher space velocities and denser feeds should be employed.

TABLE 9 Analysis of liquid products by GC-MS obtained over amorphous SiO₂/Al₂O₃ at experiments with varying feed composition and WHSV at 325° C. WHSV, h⁻¹ 1 2 1 Pressure, bar 36 36 36 Temperature, ° C. 325 325 325 Concentration of aq solution 10 10 20 of mevalonolactone, % GC-MS analysis, area % Mevalonolactone Dehydromevalonic lactone 17.55 2-methyl-2-Propanol 7.1 14.807 2-methyl-1,3-Butadiene 5.42 8.238 54.307 2-methyl-3-Buten-2-ol 3.2 3-methyl-2-Butanone 44.51 46.507 33.232 3-methyl-3-Buten-1-ol 2.31 4.982 Ethyl alcohol 2.56 5.038 2,2-dimethyl-Propanal 8.628 Ethanol, 2-(2-methoxyethoxy)- 3.45 1.3Cyclohexadiene, 3.45 1-methyl-4-(1methylethyl) Propylene Glycol 4.83 Other 5.75 11.801 12.462 Total 100.13 100.00 100.00

TABLE 10 Analysis of gaseous products obtained over amorphous SiO₂/Al₂O₃ at experiments with varying feed composition and WHSV at 325° C. WHSV, h⁻¹ 1 2 1 Pressure, bar 36 36 36 Temperature, ° C. 325 325 325 Concentration of aq solution 10 10 20 of mevalonolactone, % GC analysis, vol % Ethylene 0.007 0.008 Propane Propylene 0.007 0.012 0.01 Isobutane 0.004 1-butene 0.004 0.005 0.005 Isobutylene 1.601 1.253 Cis-2-butene 0.280 Isopentane 0.003 0.004 0.004 C₅₊ 0.045 0.092 0.104 C₆₊ 1.260 1.903 2.593 CO₂ 3.053 11.21 12.028 CO 0.063 0.154 0.128 N₂ 97.151 88.131 85.832 Total 101.973 103.119 101.965 Further analysis on experiment with 20% aq solution of mevalonolactone

As discussed above, the experiment performed with 20% aqueous solution of mevalonolactone over amorphous SiO₂/Al₂O₃ at 325° C. showed a high percentage of isoprene in the liquid product as % area of isoprene to the total product area peaks in the GC-MS analysis. As this condition demonstrated the most promising results in terms of isoprene formation, we performed further analysis of the liquid product sample in an effort to quantify the amount of isoprene. The first step comprised of measuring the amount of water in the liquid product with the Karl-Fischer method (ASTM D6304). The water was found to be ˜94 wt % in the liquid product. This indicated that the main product of the reaction was water and the concentration of organics in the liquid product sample was <6 wt %. Furthermore, we attempted to perform a semi-quantitative analysis using isoprene standard of the GC-MS to measure the actual concentration of the desired product. These results pointed out that the actual concentration of isoprene is probably <3 wt %, with the rest being 3-methyl-2-butanone and other unidentified components.

Example 14 Synthesis of Isoprene

The mevalonolactone conversion to isoprene is performed in the down-flow stainless steel fixed bed reactor loaded with amorphous SiO₂/Al₂O₃ catalyst, according to Example 11. The catalyst is pretreated in-situ at 300° C. in air for 30 min. Prior to feed introduction, the desired reaction temperature, 325 ° C., and pressure, 36 bar, is achieved under flowing inert gas (N₂). Upon reaching the desired reaction conditions, the aqueous solution of mevalonolactone (aq. 600 g mevalonolactone/L) is fed to the packed tubular reactor using a high precision pump. In order to maintain the pressure, a small N₂ flow (50 cm³/min) is co-fed with the liquid feed. The reaction is conducted at a pressure of 36 bar using a weight hourly space velocity (WHSV) of 2 h⁻¹. The WHSV is calculated based on the total liquid feed (solution). Steady-state activity measurements are taken after ˜2 hours time on-stream. Liquids are collected in a trap (˜10° C.), while gaseous samples are collected in gas sampling bags.

Additional Experiments Varying Feed Concentration and Space Velocity

Using the above described the set-up, 14.3 gm of catalyst SiO₂/Al₂O₃ (DAVICAT SIAL 3113 supplied by Grace in powder form and used as such) is loaded in the fixed bed reactor and pretreated in-situ at 300° C. in air for 30 mins. Aqueous solutions of pure Mevalonolactone obtained from Example 11 are pumped through the reactor at different conditions as summarized in Table 11. The major product in each experiment is isoprene, with 3-methyl-2-butanone as a side product.

TABLE 11 Additional Experiments varying feed concentration and space velocity Feed Space Temperature concentration velocity Pressure Condition (° C.) (%) (WHSV h⁻¹) (bar) 1 325 30% 1 36 2 325 50% 1 36 3 325 70% 1 36 4 325 30% 2 36 5 325 50% 2 36 6 325 70% 2 36 7 325 30% 3 36 8 325 50% 3 36 9 325 70% 3 36 10 325 10% 3 36

Example 15 Synthesis of Substituted Aromatics

The mevalonolactone conversion to substituted aromatics is performed in the down-flow stainless steel fixed bed reactor loaded with amorphous ZSM-5 catalyst, as described in Example 11. The catalyst is pretreated in-situ at 300° C. in air for 30 min. Prior to loading the ZSM-5 catalyst is calcined in air at 500° C. for 3 hours to concert the catalyst from the ammonium to H⁺ form. The sample is crushed and sieved to a particle size of 100-180 μm prior to use. Prior to feed introduction, the desired reaction temperature (250° C., 300° C., 350° C.), and pressure (36 bar), is achieved under flowing inert gas (N₂). Upon reaching the desired reaction conditions, the aqueous solution of mevalonolactone (aq. 10% mevalonolactone) is fed to the packed tubular reactor using a high precision pump. In order to maintain the pressure, a small N₂ flow (50 cm³/min) is co-fed with the liquid feed. The reaction is conducted at a pressure of 36 bar using a weight hourly space velocity (WHSV) of 1 h⁻¹. The WHSV is calculated based on the total liquid feed (solution). Steady-state activity measurements are taken after ˜2 hours time on-stream. Liquids are collected in a trap (˜10° C.), while gaseous samples are collected in gas sampling bags. The products are a mixture of aromatics and 3-methyl-2-butanone, as analyzed by liquid and gas chromatography

Example 16 Synthesis of Anhydro-Mevalonolactone

The mevalonolactone conversion to anhydro-mevalonolactone is performed in the down-flow stainless steel fixed bed reactor loaded with amorphous ZSM-5 catalyst, as described in Example 11. The catalyst is pretreated in-situ at 300° C. in air for 30 min. Prior to loading the ZSM-5 catalyst is calcined in air at 500° C. for 3 hours to convert the catalyst from the ammonium to H⁺ form. The sample is crushed and sieved to a particle size of 100-180 μm prior to use. Prior to feed introduction, the desired reaction temperature (70° C., 100° C., 121° C., 150° C.), and pressure (36 bar), is achieved under flowing inert gas (N₂). Upon reaching the desired reaction conditions, the aqueous solution of mevalonolactone (aq. 10% mevalonolactone) is fed to the packed tubular reactor using a high precision pump. In order to maintain the pressure, a small N₂ flow (50 cm³/min) is co-fed with the liquid feed. The reaction is conducted at a pressure of 36 bar using a weight hourly space velocity (WHSV) of 1 h⁻'. The WHSV is calculated based on the total liquid feed (solution). Steady-state activity measurements are taken after ˜2 hours on-stream. Liquids are collected in a trap (˜10° C.), while gaseous samples are collected in gas sampling bags. The major product is anhydro-mevalonolactone, as analyzed by liquid and gas chromatography

Example 17 Synthesis of Anhydro-Mevalonolactone

An acetoaceyl-CoA pool is generated by E. coli endogenously by enzyme acetyl-CoA acetyltransferase AtoB. First HMG-CoA synthase (mvaS or hmgS) and HMG-CoA reductase (mvaE or hmgR) are cloned to provide a route for the production of mevalonate from this pool. Additionally to maximize mevalonate flux, the Protein-Protein BasicLocal Alignment Search Tool (BlastP) is used to identify mvaS and mvaE from various organisms, such as Enterococcus faecalis, Staphylococcusaureus, Lactobacillus casei, Methanococcus maripaludis, and Methanococcus voltae. Combinatorial tests are used to identify the optimum set of mvaS and mvaE for mevalonate production. To scale up the production of mevalonate, the E. coli strain carrying genes from L. casei are fermented in a 1.3-Lbioreactor. To prepare anhydro-mevalonolactone, a solid acid catalyst is added directly to the fermentation broth and heated to reflux to catalyze the dehydration of mevalonate. The resulting anhydro-mevalonolactone is isolated by solvent extraction using chloroform. The combined organic phases are concentrated in vacuo to produce crude product.

Example 18 Synthesis of Anhydro-Mevalonolactone with p-Toluene Sulfonic Acid

10 gm Mevalonolactone (obtained in Example 2) was refluxed in presence of 1 gm of p-toluene sulfonic acid in toluene for 10 hrs. We observed a yield of 85% of anhydro-mevalonolactone. The product was washed with saturated NaHCO₃ and passed through silica gel plug to obtain anhydro-mevalonolactone (¹H NMR) with >95% purity.

Example 19 Synthesis of Anhydro-Mevalonolactone with Samarium Acetate

200 mg Mevalonolactone (obtained in Example 2) was bubbled with Argon gas for 30 mins in a 1 mL vial, and 5 mg samarium acetate was added under an Argon gas flow. The vial was sealed and put in a shaker at 150° C. for 48 hours Anhydro-mevalonolactone was obtained, as verified by NMR.

Example 20 Synthesis of Anhydro-Mevalonolactone with Ferric Chloride

200 mg Mevalonolactone (obtained in Example 2) was bubbled with Argon gas for 30 mins in a 1 mL vial, and 4 mg FeCl₃ was added under an Argon gas flow. The vial was sealed and put in a shaker at 150° C. for 48 hours. Anhydro-mevalonolactone was obtained, as verified by NMR.

Example 21 Synthesis of Anhydro-Mevalonolactone with Amberlyst® 35

500 mg Mevalonolactone (obtained in Example 2) was dissolved in 10 mL water and distributed equally into 10 vials. To each vial, 63 mg of Amberlyst® 35-wet (obtained from Dow) was added. After sealing the vials and pressuring them to 65psi, five vials were heated to 90° C. and 5 vials were heated to 150° C. 100 μl of samples were drawn at various time intervals, quenched and diluted with water and analyzed via LC-MS. Anhydro-mevalonolactone was the only major product formed, as highlighted in Table 12.

TABLE 12 Analysis of anhydro-mevalonolactone formation with Amberlyst ® 35 Dehydro-mevalonolactone (Area under curve, arb. Units) Time (hr) 90° C. 150° C. 1 15,077 49,471 4 16,288 58,455 8 25,710 58,300 12 26,260 70,951 24 39,171 72,303 48 49,669 69,681

Example 22 Ethenolysis of Anhydro-Mevalonolactone

An 1 L glass reactor composed of thick wall glass is charged under nitrogen or argon with an appropriate solvent, such as dichloromethane or dichloroethane (500 mL), dehydro-mevalonolactone (0.86 mol), and a Grubbs ruthenium metathesis catalyst (0.01-1.0 mol %). After stirring for 10-60 min under nitrogen, the vessel is pressurized with 150 psi of ethylene gas and the reaction is stirred at 40° C. over a period of up to 24 hours, or until process monitoring indicates that the reaction is complete. The unused ethylene is then removed and recovered, and the reaction vessel is opened to the atmosphere. Following removal of the solvent, the crude product is purified.

Example 23 Ethenolysis of 5-hydroxy-3-methyl-2-(E)-pentenoic Acid

A 1 L glass reactor composed of thick wall glass is charged under nitrogen or argon with an appropriate solvent, such as dichloromethane or dichloroethane (500 mL), 5-hydroxy-3-methyl-2-(E)-pentenoic acid (0.86 mol), and a Grubbs ruthenium metathesis catalyst (0.01-1.0 mol %). After stirring for 10-60 min under nitrogen, the vessel is pressurized with 150 psi of ethylene gas and the reaction is stirred at 40° C. over a period of up to 24 hours, or until process monitoring indicates that the reaction is complete. The unused ethylene is then removed and recovered, and the reaction vessel is opened to the atmosphere. The solution is treated with aqueous sodium hydroxide (300-500 mL, 1-5 M solution) and the aqueous layer is extracted twice with the reaction solvent. The aqueous layer is then acidified to pH 0-2, and extracted with dichloromethane or diethyl ether (5x100 mL). Following removal of the solvent, the crude product is purified.

Example 24 Methyl Ester of Anhydro-Mevalonolactone

Amberlyst® resin is added to a solution of anhydro-mevalonolactone in methanol, and the slurry is heated to reflux. After 24 h, the reaction is cooled to room temperature, filtered and concentrated in vacuo to provide crude product, which is used in further reactions without purification.

Example 25 Ethenolysis of the Methyl ester of 5-hydroxy-3-methyl-2-(Z)-pentenoic Acid

An 1 L glass reactor composed of thick wall glass is charged under nitrogen or argon with an appropriate solvent, such as dichloromethane or dichloroethane, the methyl ester of 5-hydroxy-3-methyl-2-(Z)-pentenoic acid from Example 24, and a Grubbs ruthenium metathesis catalyst (0.01-1.0 mol %). After stirring for 10-60 min under nitrogen, the vessel is pressurized with 150 psi of ethylene gas and the reaction is stirred at 40° C. over a period of up to 24 hours, or until process monitoring indicates that the reaction is complete. The unused ethylene is then removed and recovered, and the reaction vessel is opened to the atmosphere. Following removal of the solvent, the crude product is purified.

Example 26 Ethyl ester of anhydro-mevalonolactone

Amberlyst® resin is added to a solution of anhydro-mevalonolactone in ethanol, and the slurry is heated to reflux. After 24 h, the reaction is cooled to room temperature, filtered and concentrated in vacuo to provide crude product, which is used in further reactions without purification.

Example 27 Ethenolysis of the ethyl ester of 5-hydroxy-3-methyl-2-(Z)-pentenoic Acid

An 1 L glass reactor composed of thick wall glass is charged under nitrogen or argon with an appropriate solvent, such as dichloromethane or dichloroethane, the ethyl ester of 5-hydroxy-3-methyl-2-(Z)-pentenoic acid from Example 26, and a Grubbs ruthenium metathesis catalyst (0.01-1.0 mol %). After stirring for 10-60 min under nitrogen, the vessel is pressurized with 150 psi of ethylene gas and the reaction is stirred at 40° C. over a period of up to 24 hours, or until process monitoring indicates that the reaction is complete. The unused ethylene is then removed and recovered, and the reaction vessel is opened to the atmosphere. Following removal of the solvent, the crude product is purified.

Example 28 Glycerol ester of anhydro-mevalonolactone

Amberlyst® resin is added to a solution of anhydro-mevalonolactone in glycerol, and the slurry is heated to reflux. After 24 h, the reaction is cooled to room temperature, filtered and concentrated in vacuo to provide crude product, which is used in further reactions without purification.

Example 29 Ethenolysis of the glycerol ester of 5-hydroxy-3-methyl-2-(Z)-pentenoic Acid

An 1 L glass reactor composed of thick wall glass is charged under nitrogen or argon with an appropriate solvent, such as dichloromethane or dichloroethane, the glycerol ester of 5-hydroxy-3-methyl-2-(Z)-pentenoic acid from Example 28, and a Grubbs ruthenium metathesis catalyst (0.01-1.0 mol %). After stirring for 10-60 min under nitrogen, the vessel is pressurized with 150 psi of ethylene gas and the reaction is stirred at 40° C. over a period of up to 24 hours, or until process monitoring indicates that the reaction is complete. The unused ethylene is then removed and recovered, and the reaction vessel is opened to the atmosphere. Following removal of the solvent, the crude product is purified.

Example 30 Methyl ester of 5-hydroxy-3-methyl-2-(E)-pentenoic Acid

Amberlyst® resin is added to a solution of 5-hydroxy-3-methyl-2-(E)-pentenoic acid in methanol, and the slurry is heated to reflux. After 24 h, the reaction is cooled to room temperature, filtered and concentrated in vacuo to provide crude product, which is used in further reactions without purification.

Example 31 Ethenolysis of the methyl ester of 5-hydroxy-3-methyl-2-(E)-pentenoic Acid

An 1 L glass reactor composed of thick wall glass is charged under nitrogen or argon with an appropriate solvent, such as dichloromethane or dichloroethane, the methyl ester of 5-hydroxy-3-methyl-2-(E)-pentenoic acid from Example 30, and a Grubbs ruthenium metathesis catalyst (0.01-1.0 mol %). After stirring for 10-60 min under nitrogen, the vessel is pressurized with 150 psi of ethylene gas and the reaction is stirred at 40° C. over a period of up to 24 hours, or until process monitoring indicates that the reaction is complete. The unused ethylene is then removed and recovered, and the reaction vessel is opened to the atmosphere. Following removal of the solvent, the crude product is purified.

Example 32 Ethyl ester of 5-hydroxy-3-methyl-2-(E)-pentenoic Acid

Amberlyst® resin is added to a solution of 5-hydroxy-3-methyl-2-(E)-pentenoic acid in ethanol, and the slurry is heated to reflux. After 24 h, the reaction is cooled to room temperature, filtered and concentrated in vacuo to provide crude product, which is used in further reactions without purification.

Example 33 Ethenolysis of the ethyl ester of 5-hydroxy-3-methyl-2-(E)-pentenoic Acid

An 1 L glass reactor composed of thick wall glass is charged under nitrogen or argon with an appropriate solvent, such as dichloromethane or dichloroethane, the ethyl ester of 5-hydroxy-3-methyl-2-(E)-pentenoic acid from Example 32, and a Grubbs ruthenium metathesis catalyst (0.01-1.0 mol %). After stirring for 10-60 min under nitrogen, the vessel is pressurized with 150 psi of ethylene gas and the reaction is stirred at 40° C. over a period of up to 24 hours, or until process monitoring indicates that the reaction is complete. The unused ethylene is then removed and recovered, and the reaction vessel is opened to the atmosphere. Following removal of the solvent, the crude product is purified.

Example 34 Glycerol ester of 5-hydroxy-3-methyl-2-(E)-pentenoic Acid

Amberlyst® resin is added to a solution of 5-hydroxy-3-methyl-2-(E)-pentenoic acid in glycerol, and the slurry is heated to reflux. After 24 h, the reaction is cooled to room temperature, filtered and concentrated in vacuo to provide crude product, which is used in further reactions without purification.

Example 35 Ethenolysis of the glycerol ester of 5-hydroxy-3-methyl-2-(E)-pentenoic Acid

An 1 L glass reactor composed of thick wall glass is charged under nitrogen or argon with an appropriate solvent, such as dichloromethane or dichloroethane, the glycerol ester of 5-hydroxy-3-methyl-2-(E)-pentenoic acid from Example 34, and a Grubbs ruthenium metathesis catalyst (0.01-1.0 mol %). After stirring for 10-60 min under nitrogen, the vessel is pressurized with 150 psi of ethylene gas and the reaction is stirred at 40° C. over a period of up to 24 hours, or until process monitoring indicates that the reaction is complete. The unused ethylene is then removed and recovered, and the reaction vessel is opened to the atmosphere. Following removal of the solvent, the crude product is purified.

Example 36 Highly selective production of isoprene and methyl vinyl ketone with silica catalyst

A small volume fixed bed reactor was loaded with 1 gram of Davisil Grade 62 silica gel and heated to a temperature of 250 C with 1.4 sccm of nitrogen carrier gas and 2.5 uL/min flow of 20 wt % mevalonolactone in water injected via a coaxial injection. In addition, a post-reactor vapor trap held at 60-70 C is used to trap water and dehydrated mevalonolactone to prevent contamination of the in-line GC-FID used or analysis. Under these conditions, the area fraction of isoprene as analyzed using in-line GC-FID is 91% with 7% of the peak area identified as methyl vinyl ketone.

Based on peak area analysis as compared to a model stream of a molar equivalent flow of isoprene conversion is greater than 69% showing a minimum overall yield of 61% (i.e. 0.61 moles of isoprene per mole of mevalonolactone) with 91% selectivity to isoprene. In each case, by “total conversion” what is meant is the amount of mevalonolactone converted to products detectable by GC-FID within 4 minutes with a 45 degree column temperature, and explicitly does not include conversion to dehydrated mevalonolactone. It is expected that nearly 100% of mevalonolactone is consumed and converted to dehydrated mevalonolactone if it does not convert fully to other molecules.

Additional Experiments varied the residence time in the reactor, showing increased selectivity and conversion at low flow rates. At 250 C with 2.5 uL/min flow of 20 wt % mevalonolactone in water and carrier gas flow rate of 14.05 sccm showed isoprene peak area as 84% with methyl vinyl ketone as 14% of the peak area. Yield as compared to model stream suggests a total yield in excess of 48% isoprene for a minimum conversion of 56%. At an intermediate residence time with 7.01.4 sccm of carrier gas but other parameters left unchanged, the isoprene selectivity was 86% with methyl vinyl ketone as 11% of the peak area, and by the same estimation method a total conversion minimum of 67% for an overall yield of at least 59%.

Additional Experiments varied the temperature in the reactor, showing increased selectivity to isoprene at lower temperatures. At 300 C with 10 uL/min of 20% mevalonolactone in water and 28.13 sccm of nitrogen, the isoprene peak area is 72%, with estimated conversion of at least 66% for an overall yield of 47%.

Additional Experiments varied the concentration of mevalonolactone in water, using about 200 mg of Aerosil 380 silica catalyst. With 5 uL/min of 70% mevalonlactone in water and 14.05 sccm of nitrogen at 300 C, the product distribution shows 64% isoprene with 24% methyl vinyl ketone, 2% 2-methyl-1-butene, and 2% 2-methyl-1-propene. As temperature increases, the fraction of methyl vinyl ketone increases while isoprene decreases. At 350 C, isoprene is 36% of the peak area, methyl vinyl ketone is 33% of the peak area, 2-methyl-1-butene is 14% of the peak area, and 2-methyl-1-propene is 6% of the peak area. At 400 C, isoprene is 21% of the peak area, methyl vinyl ketone is 39% of the peak area, 2-methyl-1-butene is 33% of the peak area, and 2-methyl-1-propene is 6% of the peak area.

Additional Experiments removed the carrier gas and flowed 20% mevalonolactone at 2.5 uL/min into the reactor. At 250 C, the isoprene peak area was 91%, methyl vinyl ketone was 3%, and 2-methyl-1-propene was 4%. At even lower temperatures, we saw increased selectivity to isoprene. At 200 C, the isoprene peak area reached 96% with methyl vinyl ketone at 2.5%. At 150 C, the isoprene peak area reached 98.4% with methyl vinyl ketone at 1%.

Thus, by varying reactor temperature and residence time we can produce isoprene with exceptional selectivity, or switch to an intermediate condition to produce the desired product distribution of isoprene, methyl vinyl ketone, 1-methyl-2-butene, and 1-methyl-2-propene. This should not be taken as a limitation on the product distribution as our analytical tool only monitors low boiling point compounds quantitatively. Other products seen when analyzing the reactor products include but are not limited to 2-butene, 1,3-pentadiene, 2-methyl-1,3-butadiene, 2-butanone, 1,4-pentadiene-3-one, 1-pentene-3-one, 1-methyl-1,3-cyclohexadiene, 1-methyl-1,4-cyclohexadiene, toluene, 1-methylcyclohexa-2,4-diene, phenol, chlorobenzene, xylene, 4-penten-1-yl acetate, 1-(1,2-dimethyl-cyclopent-2-enyl)-ethanone, 4-acetyl-1-methylcyclohexene, and dehydrated mevalonolactone.

Example 37 Reaction of Mevalonolactone over Palladium Supported on Silica

Using the same test reactor as in EXAMPLE 36, roughly 500 mg of SiliaCat Pd0 catalyst, palladium supported on silica, was tested. At 325 C with 10 uL/min of 20 wt % MVL in water and 28.13 sccm of nitrogen carrier gas, the isoprene peak area is roughly 68% with methyl vinyl ketone at 5%, 2-methyl-1-butene at 4%, 2-methyl-1-pentene at 6%, and 2-pentene at 15%.

Additional Experiments tested different temperature ranges and conditioning processes. When the catalyst is initially held at 275 C under 10 uL/min of 20 wt % MVL in water and 28.13 sccm of nitrogen, increasing temperature results in greater selectivity to isoprene, increasing from nearly zero selectivity to 75% selectivity at 375 C with high yield. However, when the catalyst is initially held at 400 C selectivity to isoprene increases as temperature goes down to 325 C, with 40% of peak area at 375 C, 55% at 350 C, and 68% at 325 C. This is indicative of palladium producing other isomers more preferentially, with palladium slowly being deactivated. Additional Experiments tested different times on stream. As indicated in the temperature ramp direction test above, longer times on stream generally result in more palladium deactivation and higher isoprene selectivity versus other products. Other products include 2-pentene, 2-methyl-1-propene, 2-methyl-1-butene, methyl vinyl ketone, 1,3,5-tributyl-benzene, and other benzene derivatives.

Additional Experiments tested different residence times. With 5 uL/min of 20 wt % MVL in water plus 14.05 sccm of nitrogen at 325 C, isoprene area percentage is roughly 78%, with 11% for methyl vinyl ketone, 5% for 2-methyl-1-butene, and 6% for 2-methyl-1-propene. When the residence time is halved by increasing flow rate to 10 uL/min of liquid MVL solution and 28.13 sccm of nitrogen, isoprene peak area increases slightly to 82%, with 9% for methyl vinyl ketone, 3% for 2-methyl-1-butene, and 6% for 2-methyl-1-propene. When the residence time is reduced by a factor of 3, using a flow rate of 15 uL/min of MVL solution and 41.72 sccm of nitrogen, the isoprene peak area percentage decreases slightly to 81% with methyl vinyl ketone at 10%, 2-methyl-1-butene at 3%, and 2-methyl-1-propene at 7%. Based on this result, we estimate that the middle residence time is preferred for palladium supported on silica. With a roughly 1 gm/mL powder tap density, the 500 mg of catalyst has a volume of roughly 500 uL. At 325 C, lsccm of nitrogen is a real flow rate of about 2 mL/min. At 325 C, luL/min of water flow is roughly 1 mL/min of real flow. Thus, with 10 uL/min of MVL solution in water and 28.13 sccm of nitrogen, we can estimate the real flow rate as 66.26 mL/min, for an estimated residence time of 0.45 seconds.

Additional Experiments tested varying MVL concentrations in water. All tests were done at 325 C and 40 sccm of nitrogen. Initially testing 10 uL/min of 100% MVL produced isoprene with 80% of the peak area, dropping to 58% after 86 minutes for an average of about 70%. Methyl vinyl ketone was present with about 16% of the peak area, 2-methyl-1-butene with 4%, and 2-methyl-1-propene with 6%. Decreasing 100% MVL flow rate to 5 uL/min resulted in an initial isoprene peak area of about 64%, dropping to 60% over 294 minutes. Methyl vinyl ketone was present with 21% of the peak area, 2-methyl-1-butene with 7%, and 2-methyl-1-propene with 7%. Using 70 wt % MVL in water, with flow rate of 5 uL/min isoprene peak area was slightly improved with about 70% of the peak area.

Example 38 Reaction of Mevalonolactone over Alumina-Silica

Tests over alumina-silica catalyst Davicat SIAL 3113 were performed in the equipment described in EXAMPLE 36 in addition to the tests done using the reactor described in Example 11. In this followup test, similar time on-stream behavior was seen as with EXAMPLE 37 wherein initial selectivity to isoprene was nearly zero, but after many hours on stream selectivity improved. Final performance of Davicat shows 73% of peak area as isoprene with 8% as methyl isopropyl ketone. This is most distinct from other tests in that methyl isopropyl ketone is present in substantial quantities whereas other catalysts and no catalyst tests produce almost no methyl isopropyl ketone and instead favor methyl vinyl ketone formation. In addition to the list of products shown in Example 11-14, we were able to identify several isomers of 1-ethyl-n-methyl Benzene, 1,3,5-trimethyl Benzene, 1-methyl-3-(1-methylethyl) Benzene, 1,4-diethyl-Benzene, 4-ethyl-1,2-dimethyl-Benzene, 1,2,4,5-tetramethyl-Benzene, 1,2,3 ,4-tetramethyl-Benzene, 1,2,4,5-tetramethyl-Benzene, and Diethyl Phthalate. Due to strong dependence on the time-on-stream for Davicat SIAL 3113 catalyst, numbers presented should be taken as general estimates only.

Additional Experiments varying the reactor temperature show that at 300 C, 30 uL/min of 20 wt % MVL in water with 28.13 sccm of nitrogen we see isoprene peak area start at 50% and increase to 83% over 300 minutes on stream with methyl isopropyl ketone decreasing from 20% to 4% in the same time period. At 350 C, 30 uL/min of 20 wt % MVL in water with 28.13 sccm of nitrogen isoprene peak area of about 73% while methyl isopropyl ketone was about 8% of peak area.

Additional Experiments varying pressure showed that higher pressures (500 PSI) result in decreased selectivity to isoprene in favor of Benzene derivatives.

Example 39 Reaction of Mevalonolactone over Titania

Aeroxide Titania P25 was tested at temperatures ranging from 250 C to 350 C with 10 uL/min of 20 wt % MVL and 28.13 sccm of nitrogen with 500 mg of catalyst using the reactor described in EXAMPLE 36. At 350 C, isoprene represented 63% of the peak area with 13% as methyl vinyl ketone, 13% as 2-methyl-1-butene, and 8% as 2-methyl-1-propene. At 300 C, isoprene represented 68% of the peak area with 12% as methyl vinyl ketone, 8% as 2-methyl-1-butene, and 11% as 2-methyl-1-propene. At 250 C, isoprene represented 42% of the peak area with 49% as methyl vinyl ketone and 6% as 2-methyl-1-butene. Using the same conversion estimation method as described in EXAMPLE 36 using a model isoprene stream and ignoring conversion to aMVL, conversion at 250 C was at least 15% with 6% yield to isoprene. Conversion at 300 C was 44% with 30% minimum yield to isoprene. Conversion at 350 C was 55% with 35% minimum yield to isoprene.

Example 40 Reaction of Mevalonolactone without Catalyst

MVL solution was reacted with no catalyst through the reactor described in EXAMPLE 36, with roughly 3 mL of reactor internal volume heated but with no catalyst loaded at temperatures ranging from 300 C to 450 C. At all temperatures, 20 wt % MVL in water was flowed at 10 uL/min with 28.13 sccm of nitrogen carrier gas. At 300 C, isoprene represented 8% of overall peak area, 84% as methyl vinyl ketone, and 8% as 2-methyl-1-butene. Based on the model isoprene solution described in EXAMPLE 36, we estimate 11% overall conversion and 1% yield of isoprene. At 350 C, isoprene represented 12% of total peak area, with 71% as methyl vinyl ketone, 14% as 2-methyl-1-butene, and 3% as 2-methyl-1-propene. Estimated conversion is 30% with 3% isoprene yield. At 400 C, isoprene represented 14% of total peak area, with 54% as methyl vinyl ketone, 25% as 2-methyl-1-butene, and 5% as 2-methyl-1-propene. Estimated conversion is 44% with 6% yield of isoprene. At 450 C, isoprene represented 12% of total peak area, with 46% as methyl vinyl ketone, 32% as 2-methyl-1-butene, and 5% as 2-methyl-1-propene. Estimated conversion is 48% with 6% yield of isoprene. This result demonstrates clearly that without a catalyst isoprene is substantially less favorable as compared particularly to methyl vinyl ketone.

Example 41 Reaction of Mevalonolactone over Nickel Metal Supported on Alumina

MVL was reacted over 66±5% Ni catalyst on silica alumina, catalog number 31276 from Alfa Aesar using the reactor described in EXAMPLE 36. At 300 C, 10 uL/min of 20 wt % MVL and 28.13 sccm of nitrogen resulted in only one visible peak on the in-line FID after vapor trap, presumed to be methane, carbon monoxide, or hydrogen. No isoprene, methyl vinyl ketone, methyl isopropyl ketone, or other known products were identified in the product stream. Decreasing reactor temperature to 250 C resulted in no significant change to the products visible.

Example 42 Reaction of Mevalonolactone with ethanol over Silica

Using the same test reactor as in EXAMPLE 36, 1.172 gm of Davisil Grade 62 Silica Gel was loaded and held at 300 C. In this example, ethanol is added as a co-reactant by mixing 3.638 gm of 20 wt % MVL in water with an additional 0.402 gm absolute ethanol in a syringe pump which was injected at 10 uL/min into the reactor alongside 2 sccm of nitrogen. After allowing the system to equilibrate for 30 minutes, the full product distribution was collected in either dichloromethane or 1-octanol to avoid missing product peaks due to overlap with solvent. In this example, the vapor trap and in-line GC-FID are bypassed to allow full product collection including heavy and high boiling point molecules. Mass spectra for the products in dichloromethane and 1-octanol were separately captured and analyzed. Some solvent contamination is expected, and as with all mass spectra identification is always somewhat ambiguous so this should not be taken as limiting the potential products produced using ethanol or any other coreactant. The combined list of identified chemicals is

Cyclobutanol

4-Penten-2-ol, 3-methyl-

Ethanol

4-Penten-1-yl acetate

1,3-Dioxolane, 2-heptyl-4-phenyl-

Methyl vinyl ketone

Ethyl acetate

1,4-pentadien-3-one

4-pentenoic acid ethyl ester

Benzene, [(cyclohexyloxy)methyl]-

Bicyclo [2.2.2]oct-7-en-2-one, 5-methylene-

2,6-Octadien-1-ol, 2,7-dimethyl-

P-Menth-1(7)-en-9-ol

3-Cyclohexen-1-ol, 4-methyl-1-(1-methylethyl)-, acetate

2,3-Epoxycarane, (E)-

Cyclohexanol, 1-methyl-4-(1-methylethenyl)-, acetate

Ethanol, 2-(3,3-dimethylcyclohexylidene)-, (Z)-

5-Caranol, trans, trans-(+)-

2-Furanone, 2,5-dihydro-3,5-dimethyl

3-Methylene-bicyclo [3.2.1]oct-6-en-8-ol

Benzeneethanol, .alpha., .alpha.-dimethyl-, acetate

2-Cyclohexen-1-one, 4,5-dimethyl-

(3S,4R,5R,6R)-4,5-Bis(hydroxymethyl)-3,6-dimethylcyclohexene

4-Acetyl-1-methylcyclohexene

Cyclopentane, 1-acetyl-1,2-epoxy-

Ketone, 1,5-dimethylbicyclo[2.1.0]pent-5-yl methyl

Dehydromavalonic Lactone

Spiro[3.4]octan-5-one

1-Phthalanol, 1,3,3-trimethyl-

9-Octadecen-12-ynoic acid, methyl ester

2H-Pyran-2-one, 5,6-dihydro-4-(2,3-dimethyl-2-buten-2-yl)-

6-(p-Tolyl)-2-methyl-2-heptenol

4,4-Dimethylcyclohexadienone

Phenanthro[3,2-b]furan-7,11-dione, 1,2,3,4,8,9-hexahydro-4,4,8-trimethyl-, (+)-

1-Nonanol

Decanal

1-Decanol

Oxalic acid, isobutyl nonyl ester

Decanoic acid, methyl ester

Tetrahydropyran 12-tetradecyn-1-ol ether

Carbonic acid, octadecyl phenyl ester 

1. A process comprising: (a) reacting MVL, or a solution comprising MVL, in the presence of a solid catalyst to yield a first product or first product mixture.
 2. The process of claim 1, wherein the solution comprising MVL comprises anhydro-mevalonolactone.
 3. The process of claim 1, wherein the solution comprising MVL comprises a co-reactant.
 4. The process of claim 3, wherein the co-reactant is selected from the group consisting of water, alkyl alcohol, ethers, aromatic compounds, acids, aldehydes, esters, and mixtures thereof.
 5. The process of claim 1, wherein step (a) occurs at a temperature in the range of about 100° C. to about 500° C.
 6. The process of claim 1, wherein step (a) occurs at a pressure in the range of about 10 bar to about 100 bar.
 7. The process of claim 1, wherein step (a) occurs at a temperature in the range of about 100° C. to about 500° C. and at a pressure in the range of about 10 bar to about 100 bar.
 8. The process of claim 1, wherein said solid catalyst is a solid acid catalyst.
 9. The process of claim 8, wherein said solid catalyst is selected from the group consisting of functionalized styrene-divinylbenzene copolymers, functionalized tetrafluoroethylene-fluoropolymer copolymers, calcium apatite, silica-alumina, silica, titania, alumina, resins, metal oxides, zeolites, and combinations thereof.
 10. The process of claim 1, wherein said solid catalyst is selected from the group consisting of a metal oxide catalyst and a carbon catalyst.
 11. The process of claim 10, wherein said solid catalyst has a surface area between about 20 g/m² and about 600 g/m².
 12. The process of claim 10, wherein said solid catalyst is selected from the group consisting of silicon oxide, aluminum oxide, titanium oxide, magnesium oxide, zirconium oxide, cobalt oxide, iron oxide, nickel oxide, manganese oxide, zinc oxide, molybdenum oxide, tungsten oxide, calcium oxide, cerium oxide, tin oxide, and chromium oxide, and mixtures thereof.
 13. The process of claim 10, wherein said solid catalyst comprises a mixed metal oxide catalyst.
 14. The process of claim 1, wherein the solid catalyst comprises a supported transition or noble metal.
 15. The process of claim 14, wherein the supported transition or noble metal is selected from the group consisting of magnesium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, zirconium, molybdenum, ruthenium, rhodium, silver, tin, tungsten, tantalum, iridium, platinum, and gold, and mixtures thereof.
 16. The process of claim 1, wherein said first product or first product mixture comprises one or more of isoprene, 3-methyl-2-butanone, methyl vinyl ketone, anhydro-mevalonolactone, 2-pentene, 2-methyl-1-butene, 2-methyl-1-pentene, methane, hydrogen, or an alkyl alcohol.
 17. The process of claim 1, further comprising a step of: (b) reacting said first product or first product mixture with a first reagent and a second catalyst to yield a second product or second product mixture, wherein said first reagent is an olefin or an alcohol.
 18. The process of claim 17, wherein said first reagent is an olefin and said second catalyst is selected from the group consisting of ruthenium, molybdenum, tungsten, rhenium, tin, and rhodium olefin metathesis catalysts, and mixtures thereof.
 19. The process of claim 17, wherein said first reagent is an alcohol and said second catalyst is selected from the group consisting of homogenous and heterogeneous acids and bases, sugars, lipases, ion exchange resins, metal oxides, and zeolites, and mixtures thereof.
 20. The process of claim 17, wherein step (b) occurs at a temperature in the range of about 100° C. to about 500° C.
 21. The process of claim 17, further comprising: (c) reacting said second product or second product mixture with a second reagent and a third catalyst to yield a third product or third product mixture.
 22. A process comprising: (a) reacting MVL, or a solution comprising MVL, at a temperature in the range of about 100° C. to about 500° C. to yield a first product or first product mixture.
 23. A process comprising: (a) providing a microbial organism that expresses a biosynthetic MVL pathway; (b) growing the microbial organism in fermentation medium comprising at least one carbon substrate, whereby biobased MVL is produced; and (c) reacting said biobased MVL to yield a first product or first product mixture.
 24. The process of claim 23, wherein said step (c) occurs in the presence of a solid catalyst.
 25. The process of claim 23, wherein said step (c) occurs at a temperature in the range of about 100° C. to about 500° C.
 26. The process of claim 23, wherein said microbial organism is a non-naturally occurring microbial organism comprising a metabolic modification that enables biobased MVL production.
 27. The process of claim 26, wherein said metabolic modifications comprises expression of at least one gene set selected from the group consisting of: i. mvaE, mvaS, mvaE, mvaS, atoB, atoB, hmgR, hmgS, and functional homologs of i-iii.
 28. The process of claim 26, wherein said metabolic modification comprises modification of at least one gene.
 29. The process of claim 28, wherein said gene is selected from the group consisting of ackA-pta, adhE, and ldhA.
 30. The process of claim 23, wherein said fermentation medium comprises a renewable feedstock. 